Monoclonal antibodies and cell surface antigens for the detection and treatment of small cell lung cancer (SCLC)

ABSTRACT

The invention provides new monoclonal antibodies and binding fragments thereof which recognize and immunoreact with cell surface antigens found on small cell lung cancer (SCLC) cells. The antibodies have tumor specificity and are useful for therapy, diagnosis, monitoring, detecting and imaging of SCLC disease and of patients having SCLC disease. The antibody-recognized SCLC-specific surface antigens can serve as targets for detecting, diagnosing, inhibiting or killing SCLC cells.

[0001] This application claims benefit of provisional application U.S.Ser. No. 60/245,340, filed Nov. 2, 2000.

FIELD OF THE INVENTION

[0002] The present invention relates generally to antibodies and morespecifically to monoclonal antibodies (MoAbs), having specificity forparticular cancer cells and types, as well as to methods for detecting,diagnosing, monitoring, staging, imaging and/or treating cancers, inparticular, small cell lung cancer. The invention further relates to theantigens comprising epitopes on the surfaces of small cell lung cancercells that are recognized by the MoAbs of the present invention.

BACKGROUND OF THE INVENTION

[0003] Small Cell Lung Cancer (SCLC), an aggressive form of lung cancer,represents about 20% of primary lung tumors and exhibits the mostmalignant phenotype of lung cancer (reviewed in Livingston, R. B., 1997,“Combined modality therapy of lung cancer”, Clin Cancer Res., 3:2638-47and Gazdar, A. F., 1994, “The molecular and cellular basis of human lungcancer”, Anticancer Res., 13:261-68). Two thirds of patients diagnosedwith SCLC are between the ages of 50 and 70, with a huge, althoughdeclining, male preponderance. There is no evidence for geneticpredisposition linked with SCLC.

[0004] The predominant risk factor for SCLC is cigarette smoking. Morethan 95% of patients with SCLC are current or past smokers with directcorrelation to both the number of cigarettes smoked per day and theduration of smoking. Ionizing radiation and occupational carcinogens areadditional known risk factors for SCLC.

[0005] The natural history of SCLC differs from other types of lungcancer in the early and extensive spread of the disease. Seventy toeighty percent of the patients with SCLC have dissemination, occult orotherwise, at the time of presentation. Furthermore, SCLC has a rapidgrowth rate and the fastest doubling time of all of the types of lungcancer (25-160 days). The median survival time from the time ofdiagnosis is about 1.5 to 3 months for patients with extensive orlimited disease, respectively.

[0006] SCLC usually presents as large, rapidly developing lesionsarising from the centrally located tracheobronchial airways and invadingthe mediastinum (Stahel, R. A. et al., 1989, “Staging and prognosticfactors in small cell lung cancer: A consensus report”, Lung Cancer,5:119-26). Typically, patients present with a cough or dyspnea,wheezing, and/or chest pain. Weight loss, fatigue and anorexia occur inup to one third of the patients. At the time of diagnosis, two thirds ofthe patients with SCLC have one or more clinically detectable distantmetastases, including bone (30%), liver (25%), bone marrow (20%) and thecentral nervous system (10%). In a screening setting, sensitivity ofX-ray ranges from 45-50%, sputum cytology from 25-30% and theircombination amounts to about 60-70% specificity of positive diagnosis.

[0007] SCLC is a form of lung cancer characterized by a neuroendocrinephenotype. This is evidenced by the presence of neurosecretory granulesvisible ultrastructurally in the cytoplasm. These granules containpeptide hormones such as ADH, gastrin releasing peptide and neuromedin.The neural cell adhesion molecule is found on the cell surface. Othercell surface antigens are also linked with other forms of lung cancer.Due to their low specificity and sensitivity, tumor markers, likeneuron-specific enolase (NSE), creatinin kinase BB, orneuro-endocrinological markers, are not useful in the diagnostic phaseof SCLC disease. The above tumor markers are elevated in about 60-65% ofcases at the time of diagnosis of SCLC and correlate with tumor bulk.The usefulness of “relapse diagnosis anticipation”, i.e., the attempt tocorrelate the level of tumor markers with tumor progression and disease,is marginal since salvage treatments are virtually non-existent.

[0008] Since most SCLC patients are not candidates for surgery, thestandard treatment for SCLC includes chemotherapy and radiotherapy instages I-IV. SCLC is highly sensitive to initial chemotherapy. In spiteof this early responsiveness, residual cells inflict a fatal relapse inmost patients due to a re-emergence of chemoresistant variants.Consequently, since the cure rate is extremely low for patients withextensive SCLC disease, treatment must be considered palliative. Forpatients with relapsed, progressive disease, chemotherapy seldom showsclinical effectiveness or provides a lasting response.

[0009] More than 100 years ago, Paul Ehrlich proposed the use ofantibodies as “magic bullets” to deliver toxins to cancer cells. Thepotential of targeted immunotherapy has since attracted the attention ofgenerations of investigators. In 1975, with the development of thetechnology for producing monoclonal antibodies (MoAbs), (G. Kohler andC. Milstein, 1975, Nature, 256:495-497), it seemed that successfulantibody therapy was imminent. However, early trials with monoclonalantibodies revealed significant obstacles to their use in cancertherapy. For example, immune rejection of murine monoclonal antibodiesconstituted the primary hurdle for making antibody therapy an effectiveand successful therapeutic. In addition, disappointingly low levels ofcytotoxicity were reported during initial clinical experience (L. W.Kwak et al., 1995, Clinical applications of monoclonal antibodies, In:Biologic Therapy of Cancer, Eds. V. T. DeVita, Jr., S. Hellman and S. A.Rosenberg, 2nd Ed., J. B. Lippincott Co., Philadelphia, Pa., pp.553-565).

[0010] Experience to date suggests that only a small fraction ofinjected antibody actually reaches a tumor (R. A. Miller et al., 1981,Lancet, ii:226-230). To maximize antibody binding to target molecules,an ideal antibody for cancer therapy should have a high affinity for itsantigen (A. Hekman et al., 1991, Cancer Immunol. Immunother.,32:364-372). In addition, an effective unconjugated antibody should worksynergistically with the host's immune system effector mechanisms.Therapeutic antibodies that induce effector mechanisms such asantibody-dependent cellular cytotoxicity (ADCC) and complement-dependentcytolysis (T. A. Waldman et al., 1994, Ann. Oncol, 5 Suppl. 1:13-17)have the potential to provide targeted cancer therapy that is safe andeffective without the use of potentially harmful conjugates such astoxins or radionuclides.

[0011] Nearly all monoclonal antibodies recognizing antigens on humancancer cells also bind to normal human cells expressing the same antigen(J. G. Jurcic et al., 1996, Cancer Chemotherapy and Biological ResponseModifiers Annual, Eds. H. M. Pinedo, D. L. Longo and B. A. Chabner, pp.168-188). This cross-reactivity potentially compromises therapeuticeffectiveness and raises issues of toxicity, leading to the continuedinterest in defining antigenic targets that are unique to tumor cells.

[0012] Human SCLC is considered to be a feasible target forimmunotherapy using radiolabeled monoclonal antibodies (J. Zeuthen andA. J. Vangsted, 1993, Acta Oncologica, 32:845-51; Y. Olabiran et al.,1994, Br. J. Cancer, 69:247-52; A. Smith et al., 1991, Oncology,64:263-6; P. L. Beaumier et al., 1991, Cancer Res., 51:676-81; R. A.Stahel, 1989, Chest, 96:27S-29S; and M. Hosono et al., 1994, J. NuclearMed., 35:296-300). However, SCLC-specific MoAbs isolated to date alsoreact against one or more of neuroendocrine tissues, unrelated cells, ornormal immune cells. Examples include MoAbs binding to neuronal celladhesion molecule (NCAM), polysialic acid, cluster w4 antigen (CD24) (D.Jackson et al., 1992, Cancer Res., 52:5264-70), sialoglycoproteinantigen sGP 90-135, ganglioside GD2, and ganglioside fucosyl-GM1(FucGM1) (J. Zeuthen and A. J. Vangsted, 1993, Acta Oncologica,32:845-51). A MoAb (N901), which is specific to NCAM (CD56), wasreported to bind to SCLC tumors and cell lines, as well as to cardiacmuscles, natural killer (NK) cells, and peripheral nerves.

[0013] Understanding the tissue distribution of tumor-associatedantigens on cancers and normal tissues is essential for the selection oftargets for cancer immunotherapy. The majority of cancer antigens areself-antigens that are derived from and expressed by normal cells.Frequently, the cancer antigen is identical to the normal antigenalthough it is expressed at higher levels or endowed with a negligiblemutation insufficient for its distinction from the self-antigen. One ofthe escape mechanisms of malignant cells from the immune system is theirresemblance to their normal cell counterparts, thereby resulting in lowvisibility for the malignant cells by an individual's immunesurveillance system.

[0014] The process that leads to the discovery of unique cancer antigensis long, tedious and elaborate. The screening process entails anexhaustive weeding out of antigens expressed on both cancer or tumorcells and normal tissues. The probes used for the discovery of suchantigens are limited in their efficacy due to the fundamentally lowimmunogenicity of tumor antigens. In addition, serum samples with hightiters from cancer patients are generally scarce. Utilization of suchprobes for screening is frequently thwarted due to the “identification”of multiple artifacts, or to false-positive hits.

[0015] Antigens are diverse in their immunogenicity, i.e. their abilityto stimulate the immune response. When several antigens possessingdistinct immunogenic properties co-exist in an antigenic preparation,their antigenic dominance regulates the intensity of the immune responseto antigens. Therefore, the most robust immune response is developedagainst the strongest epitopes found in the antigenic preparation.Antigens with weaker immunogenicity will be disregarded, or the level ofthe immune response elicited against them will be negligible, due to thefocus of the immune response on the stronger and more dominant epitopes.Hence, minor epitopes contained in an antigenic preparation (e.g., avaccine) will be masked by the more immunogenic epitopes. Generatingdiscerning MoAbs against a repertoire of minor epitopes present in anantigenic preparation (such as subcellular fractions of cancer cells)containing dominant epitopes has remained a challenge for many years.

[0016] However, in spite of the above-mentioned obstacles, the presentinvention provides new and specific monoclonal antibodies which areimmunoreactive with SCLC cell surface antigens and which are useful inimmunotherapy, diagnostic, imaging, monitoring and screeningmethodologies, to name a few. The present invention has solved theproblem of generating myriads of non-specific antibodies by employing atechnique of differential immunization, which involves, in part,tolerization with closely related antigens, e.g., on normal cells or ontumor cells exhibiting a similar phenotype, followed by immunizationwith the neoplastic cells of interest having unique cell surface antigenmolecules. This differential/tolerization process allows the weeding outof B cells possessing undesired specificities from the entire pool of Bcells, prior to the fusion of the B cells with immortalized cells tocreate hybridomas. Consequently, the frequency of hybridomas with thesought-after antigenic specificities is amplified in accordance with thepresent invention, and the entire screening process is greatlysimplified.

SUMMARY OF THE INVENTION

[0017] It is an object of the present invention to provide monoclonalantibodies, or binding fragments thereof, which specifically bind toepitopes of antigen molecules present on small cell lung cancer (orcancer) cells. In accordance with the present invention, the monoclonalantibodies (specifically, e.g., MoAbs 51.2, 109.12 and 37.14) detect andbind to a single chain glycoprotein antigen having a molecular weight(MW) of about 200 kilodaltons (KDa), as determined by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) under reducingconditions. In addition, MoAb 37.14 reacts with an antigen comprisingtwo polypeptide chains, closely related in mass, and having a molecularweight of between about 35-50 KDa, thus indicating its ability toimmunoreact with another epitopic site on the SCLC antigen. Yet anotherof the monoclonal antibodies (e.g., MoAb 26.1) binds to a SCLC-specificmolecule which likely comprises a conformational epitope of a SCLCsurface antigen.

[0018] Also, according to the present invention, the anti-SCLCantibodies preferably do not bind to any appreciable extent to antigenspresent on human neuroendocrine cell lines (e.g., neuroblastoma), or toantigens present on human multiple myeloma (MM) cells (e.g., RPMI 8226and U266), or to antigens present on normal cells (e.g., normal lungcells). In particular, representative monoclonal antibodies of thepresent invention having high specificity to antigenic epitopes presenton surface molecules of SCLC cells are identified herein as MoAbsIMM010.26.1 (26.1), IMM010.141.7 (141.7), IMM010.92.7 (92.7),IMM010.37.14 (37.14), IMM010.51.2 (51.2), IMM010.109.12 (109.12),IMM010.26.5 (26.5), IMM010.106.3 (106.3), IMM010.43.7 (43.7),IMM010.142.1 (142.1), IMM010.37.26 (37.26) and IMM010.21.7 (21.7).

[0019] Another object of the present invention relates to antibodiesthat are capable of binding to the same antigenic determinant on SCLC asdo the monoclonal antibodies described herein, particularly, themonoclonal antibodies produced by the hybridoma cell lines depositedwith the American Type Culture Collection (ATCC), 10801 UniversityBoulevard, Manassas, Va., 20110-2209, on Aug. 10, 2000 and having ATCCAccession Nos. PTA-2360 and PTA-2357 (51.2 and 109.12, respectively),and to binding fragments of a monoclonal antibody capable of binding thesame antigenic determinants as the monoclonal antibodies describedherein, particularly, the monoclonal antibodies produced by thehybridoma cell lines deposited with the American Type Culture Collectionhaving ATCC Accession Nos. PTA-2360 and PTA-2357 (MoAbs 51.2 and 109.12,respectively).

[0020] Another object of the present invention relates to antibodiesthat are capable of binding to the same antigenic determinant on SCLC asdo the monoclonal antibodies described herein, particularly, themonoclonal antibody produced by the hybridoma cell line deposited withthe American Type Culture Collection (ATCC) on Aug. 10, 2000 and havingATCC Accession No. PTA-2358 (MoAb 37.14), and to binding fragments of amonoclonal antibody capable of binding to the same antigenicdeterminants as the monoclonal antibody described herein, particularly,the monoclonal antibody produced by the hybridoma cell line depositedwith the American Type Culture Collection (ATCC) and having ATCCAccession No. PTA-2358 (MoAb 37.14).

[0021] It is another object of the present invention to provide ahybridoma cell line, produced by hybridoma technology as known in theart, which produces a monoclonal antibody that specifically binds toepitopes on surface antigens on SCLC cells. The antigens recognized bythe antibodies of this invention comprise a single chain glycoproteinantigen having a molecular weight (MW) of about 200 kilodaltons (KDa),as determined by SDS-PAGE under reducing conditions, and an antigen,possibly a breakdown product of the 200 KDa antigen, comprising twopolypeptide chains, closely related in mass, and having a molecularweight of between about 35-50 KDa, as determined by SDS-PAGE underreducing conditions. Nonlimiting examples of clonally derived hybridomacell lines include the subcloned monoclonal antibody-producing hybridomacell lines designated 51.2, 109.12 and 37.14. Also in accordance withthe present invention are monoclonal antibodies that recognizeconformational epitopes on SCLC cell surface antigen molecules, such asthe 26.1 monoclonal antibody described herein.

[0022] Another object of the present invention is to provide hybridomacell lines deposited at the American Type Culture Collection (ATCC) onAug. 10, 2000, and designated as IMM010.109.12 (109.12), ATCCDesignation No. PTA-2357; IMM010.37.14 (37.14), ATCC Designation No.PTA-2358; IMM010.26.1 (26.1), ATCC Designation No. PTA-2359; andIMM010.51.2 (51.2), ATCC Designation No. PTA-2360.

[0023] It is yet another object of the present invention to provideisolated cell surface glycoprotein antigens of SCLC comprising epitopesthat are recognized and bound by the monoclonal antibodies describedherein. In accordance with the present invention, these antigens are notpresent, or are present in significantly lower amounts, preferably atundetectable levels, on normal human cells, human multiple myelomacells, or neuroendocrine cells of related origin. In addition, theantigens comprise (i) a single glycosylated polypeptide having amolecular weight of about 200 KDa, as well as (ii) an antigen moleculehaving two polypeptide chains of similar molecular mass and being in amolecular weight range of between about 35-50 KDa. The properties ofthese recognized antigens were determined by SDS-PAGE under reducingconditions. The isolated SCLC surface antigen according to (i) above isspecifically recognized by monoclonal antibodies 51.2, 109.12 and 37.14as described herein; the isolated SCLC surface antigen according to (ii)above is specifically recognized by monoclonal antibody 37.14 herein.

[0024] Yet another object of the present invention is to provide methodsof inhibiting or killing SCLC in an individual afflicted with SCLC, byadministering one or more of the monoclonal antibodies, for example,either alone, or together with others of the antibodies in a cocktail,or one or more binding fragments thereof, or administering a mixture ofintact antibody(ies) and binding fragment(s) thereof, under conditionssufficient for the binding of the one or more monoclonal antibodies, orbinding fragment thereof, to the SCLC cells, so as to result in theinhibition or the killing of the cancer cells by the immune cells of theindividual. In accordance with one embodiment of the present invention,the monoclonal antibody, or binding fragment thereof, is conjugated to atoxic or a cytotoxic moiety, such as a chemotherapeutic agent, aphotoactivated toxin, or a radioactive agent as described herein.

[0025] It is a further object of the present invention to provide aconjugate of the monoclonal antibody, or binding fragment thereof, and atoxic or cytotoxic moiety, for killing or inhibiting SCLC cells.

[0026] Another object of the present invention is to provideanti-idiotypic antibodies that mirror the binding site(s) of themonoclonal antibodies according to the present invention, and which arespecific for SCLC epitopes, e.g., conformational epitopes, recognized bythe antibodies of this invention. Such anti-idiotypic antibodies may beused for the treatment of SCLC by active immunization.

[0027] It is another object of the present invention to providemonoclonal antibodies having specificity to SCLC, or binding fragmentsthereof, bound to a solid support, substrate or matrix. Also, accordingto the present invention, the described monoclonal antibodies, orbinding fragments thereof, can be labeled with a detectable label, suchas a fluorophore, a chromophore, a radionuclide, or an enzyme, for useas diagnostic, therapeutic, imaging, and screening compounds, forexample.

[0028] It is yet another object of the present invention to provide amethod for localizing SCLC in a patient by administering one or more ofthe monoclonal antibodies described herein, or binding fragmentsthereof, to bind to the cancer cells or tumor cells within the patientand determining the location of the one or more monoclonal antibodies,or binding fragments thereof, within the patient. For such methods, themonoclonal antibodies, or binding fragments thereof, are preferablylabeled with a detectable and physiologically acceptable label, such asa radionuclide.

[0029] A further object of the present invention provides the detectionof the cell surface antigens, e.g., glycoproteins, as described hereinin a body fluid sample, to aid in the diagnosis of SCLC, or other cellsexpressing an epitope recognized by one or more of the antibodiesherein, by detecting the antigen shed, sloughed off, or released fromthe cancer or tumor cells into a body fluid, for example, in blood orserum. In addition, according to the present invention, the stage of thedisease, and/or the effectiveness of anticancer therapies, can bemonitored by determining the levels or changes over time of thespecifically recognized SCLC surface antigen in the body fluid.

[0030] It is another object of the present invention to providepharmaceutical compositions comprising one or more of the monoclonalantibodies, or binding fragments thereof, and a pharmaceuticallyacceptable carrier, diluent, or excipient.

[0031] Further objects and advantages afforded by the present inventionwill be apparent from the detailed description hereinbelow.

DESCRIPTION OF THE DRAWINGS

[0032] The appended drawings of the figures are presented to furtherdescribe the invention and to assist in its understanding throughclarification of its various aspects.

[0033]FIG. 1 presents the results of an ELISA immunoassay whichdemonstrates that the monoclonal antibodies as described herein exhibithigh binding specificity to cell surface antigens present on SCLC cells.

[0034]FIG. 2 shows the results of a scan of a Western Blot performedunder reducing conditions to assess the specificity of the monoclonalantibody 51.2 (MoAb 51.2) for SCLC antigen. All lanes contained an equaltotal concentration of membrane preparation from the designated celllines. The membrane preparation is loaded on an SDS gel in an amount of50 micrograms per lane. Lane 1: SCLC cell membranes; lane 2:neuroblastoma cell membranes; lane 3: multiple myeloma (MM) cellmembranes. As shown in FIG. 2, MoAb 51.2 detected an antigen presentmost significantly on SCLC cells and having a MW of about 200 KDa. Theappearance of a single fuzzy band indicates that the molecule recognizedby MoAb 51.2 is a single chain glycoprotein. A faint band appeared inthe neuroblastoma lane 2, which contained cell membranes from a pool ofneuroendocrine cells (i.e., SK-N-AS, MC-IXC and Be(2)-M17), but wasabsent in the myeloma (MM) lane 3, which consisted of cell membranesfrom a pool of MM cells (RPMI 8226 and U266). This result may indicatethat the recognized antigen is over-expressed or amplified in the SCLCcells, compared with the closely-related neuroblastoma cells, which areneuroendocrine in origin. MM cells, which are more developmentallydistant from SCLC cells than are neuroblastoma cells, do not appear toexpress the recognized antigen.

[0035]FIG. 3 shows the results of a scan of a Western Blot performedunder reducing conditions to assess the specificity of the monoclonalantibody 109.12 (MoAb 109.12) for SCLC antigen. All lanes contained anequal total concentration of membrane preparation from the designatedcell lines. Lane 1: multiple myeloma (MM) cell membranes; lane 2:neuroblastoma cell membranes; lane 3: SCLC cell membranes. As shown inFIG. 3, MoAb 109.12 detected an antigen present significantly on SCLCcells and having a MW of about 200 KDa. The appearance of a single fuzzyband indicates that the molecule recognized by MoAb 109.12 is a singlechain glycoprotein. The control membrane preparations containing cellmembrane s from a pool of neuroendocrine cells (i.e., SK-N-AS, MC-IXCand Be(2)-M 17), lane 2, and cell membranes from a pool of MM cells(i.e., RPMI 8226 and U266), lane 1, were not stained by MoAb 109.12.

[0036]FIG. 4 shows the results of a scan of a Western Blot performedunder reducing conditions to assess the specificity of the monoclonalantibody 37.14 (MoAb 37.14) for SCLC antigen. All lanes contained anequal total concentration of membrane preparation from the designatedcell lines. Lane 1: SCLC cell membranes; lane 2: neuroblastoma cellmembranes; lane 3: multiple myeloma (MM) cell membranes. As shown inFIG. 4, MoAb 37.14 detected an SCLC antigen having a MW in the range ofabout 35-50 KDa. This antigen appeared to consist of two fragments orchains, which were closely related in mass. The control membranepreparations from a pool of neuroendocrine cells (i.e., SK-N-AS, MC-IXCand Be(2)-M 17), lane 2, and from a pool of MM cells (i.e., RPMI 8226and U266), lane 1, were not stained by MoAb 37.14.

[0037]FIG. 5 shows a fluorescence activated cell sorter (FACS) analysisof the reactivity of the SCLC-specific monoclonal antibody 26.1,generated according to the present invention, against antigens presenton SCLC cells, neuroblastoma cells and multiple myeloma cells.Supernatant containing monoclonal antibody from the (IMM010) 26.1hybridoma clone was assayed for antibody reactivity with the variouscell types. A fluorescein isothiocyanate (FITC)-labeled anti-mouse IgGwas used to detect the presence of MAb 21.6 bound to cells.

[0038]FIGS. 6A and 6B present the results of tumor specificity studiesemploying a representative anti-SCLC antibody according to the presentinvention. In FIG. 6A, membranes prepared from human SCLC tissue(NCI-H209), human neuroendocrine tissue (Be(2)-M17), human (non-SCLC)lung tissue (NCI-H2106), human multiple myeloma tissue (RPMI-8226),human pancreatic tissue (HPAC), human ovarian tissues (fresh ovariantissue and MDAH-2774), human chronic myelogenous leukemia tissue (K562)and a human B cell tumor (Namalwa) were fractionated on a SDS-gel(4-20%). The gel was blotted onto nitrocellulose and incubated with therepresentative IMM010.109.12 MoAb. In FIG. 6B, membranes prepared fromhuman SCLC tissue (NCI-H209), human pancreatic tissues (HPAC, BxPc-3 andMia-PaCa2), human B cell tumors (IM9 and HT), human breast tissues(fresh breast tissue and T-47D) and human prostate tissue (LnCap) werefractionated on a SDS-gel (4-20%). The gel was blotted ontonitrocellulose and incubated with IMMOI0.109.12 MoAb of the presentinvention.

[0039]FIGS. 7A and 7B present the results of internalization studiesusing MoAb 51.2 of the present invention. FIG. 7A: Intracellularstaining of human neuroendocrine cancer cells (Be(2)-M17) with MoAbIMM010.51.2 (MoAb 51.2). Cells were stained extracellularly withIMM010.51.2. After fixation and permeabilization, cells were stainedintracellularly with phycoerythrin (PE)-conjugated monoclonal antibodyor a PE-conjugated isotype control. In the histograms, the solid, filledin area represents the amount of staining detected by the isotypecontrol antibody. The darker, thicker line represents the amount ofstaining detected by the anti-mouse Ig antibody. FIG. 7B: Intracellularstaining of human SCLC cells (NCI-H209) with MoAb IMM010.51.2. Cellswere stained extracellularly with IMM010.51.2. After fixation andpermeabilization, cells were stained intracellularly with aPE-conjugated Ig antibody or a PE-conjugated isotype control. In thehistograms, the solid, filled in area represents the amount of stainingdetected by the isotype control antibody. The darker, thicker linerepresents the amount of staining detected by the anti-mouse lgantibody.

[0040]FIGS. 8A and 8B shows that monoclonal antibody 109 (MoAb 109.12)identifies a 2-subunit SCLS-antigen of lower MW following treatment ofSCLC cell membranes with detergent. Cell membranes were extracted fromSCLC cultures of NCI-H209 cell lines (Example 1). Part of the membranepreparation was solubilized with NP-40 (2%). The membrane preparationswere fractionated on an SDS gradient gel (4-20%), which was blotted ontonitrocellulose and incubated with MoAb 109.12. FIG. 8A depicts theprotein scan of fractionated SCLC cell membranes following staining ofthe gel with Coomasie Blue. FIG. 8B depicts a scan of Western Blots ofSCLC cell membrane, unsolubilized (i.e., (−) Detergent) compared withsolubilized (i.e., (+) Detergent) with NP-40 and incubated with MoAb109.12. Detergent treatment of SCLC membranes was shown to convert thesingle chain, high MW approximately 200 KDa antigen into a two-subunit,lower MW antigen as shown in FIG. 8B.

[0041] FIGS. 9A-9D illustrate the results of an immunohistochemicalstudy using a representative monoclonal antibody according to thepresent invention, MoAb 51.2, versus an isotype-matched controlmonoclonal antibody on clinical samples. The results indicate thespecific and preferential immunoreactivity of 51.2 MoAb with canceroustissue excised from SCLC patients compared with the lack ofimmunoreactivity with normal human lung sections. FIG. 9A shows theresult of the control MoAb used to stain normal human lung tissue; FIG.9B shows the result of MoAb 51.2 staining of normal human lung tissue.FIG. 9C shows the result of control MoAb staining of human SCLC tissue;FIG. 9D shows the result of MoAb 51.2 staining of human SCLC tissue.

DETAILED DESCRIPTION OF THE INVENTION

[0042] The identification of unique cancer antigens enables the designof selective immunotherapy for neoplastic diseases. The capacity toutilize a determinant that is exclusively expressed by cancer cells ortumor cells, but that is not present in normal cells and tissues,insures the targeting and elimination of the neoplastic cells, whileinsulating the viability and function of the normal cells. For manymalignancies and carcinomas, novel cancer antigens have not beendefined, and antibodies immunoreactive with specific cancer antigenshave not been found.

[0043] The majority of cancer antigens are self-antigens, orauto-antigens, that are derived from and expressed by normal cells.Frequently, the cancer antigen is identical to the normal counterpartcell antigen, although it is expressed at higher levels, or is endowedwith a negligible mutation that is not sufficient to allow it to bedistinguished from the self-antigen. One of the escape mechanisms ofmalignant cells from the surveillance of the immune system is thegeneral phenotypic similarity of the malignant or cancer cells to theirnormal cell counterparts, thus resulting in the low visibility of themalignant or cancer cells in the immune system and their general escapefrom eradication by the immune surveillance cells.

[0044] The present invention provides monoclonal antibodies thatspecifically recognize and bind to epitopes on cell surface antigensexpressed by tumor cells or cancer cells of small cell lung carcinoma orcancer (SCLC). A number of the monoclonal antibodies described hereinhave been found to recognize and bind to surface glycoprotein moleculesthat are either exclusively present, or highly expressed, on SCLC cells,but which are absent from, or less highly expressed or displayed, ondevelopmentally related neuroendocrine cells which serve as controls. Inaddition, the SCLC-specific antigens recognized by the monoclonalantibodies described herein are absent from or undetectable on unrelatedcancer cells, such as multiple myeloma cells. The newly-discoveredSCLC-specific surface antigens provide targets for therapeuticintervention in SCLC disease, as well as for diagnostic and cellpurification purposes.

[0045] In one aspect of the present invention, the use of a differentialimmunization protocol allows the focusing of the humoral immune responsein an immunized animal host (including humans) to cancer antigensuniquely expressed on SCLC cells. The immune response by the host isrobust and results in the elicitation of enhanced concentrations ofserum antibodies having superior affinity to immunogen. In this way,monoclonal antibodies having specificity and high affinity for SCLCsurface antigens can be produced and used in compositions for treatmentsand therapies for patients having SCLC disease, as well as in diagnosticand/or screening protocols for determining and/or monitoring disease.

[0046] In another aspect of the present invention, a technique known ascontrasting immunization is used to obtain monoclonal antibodies toantigen, in particular, SCLC-specific antigen, and to identify the SCLCantigens described herein. Two divergent immunogens provided atdifferent anatomical locations are used (e.g., Example 1). The dualimmunization polarizes the migration of the distinct populations ofimmune cells to discrete draining lymph nodes. For example, a pool ofhuman SCLC cell lines is used as the immunogen to obtain murinemonoclonal antibodies to an antigen specific to SCLC. A high dose ofcontrol cells, which constitute a pool of related neuroendocrine celllines, is used to polarize the immune response, so as to effectivelydelete undesired immune cells from the lymph nodes near to the site ofimmunization with the desired antigen. The immune cells extracted fromthe draining lymph nodes close to the site of immunization with thedesired SCLC cell immunogens are then immortalized by fusion with murinemyeloma cells, using hybridoma protocols known and practiced in the art.Following this type of immunization protocol, for example, the antipodaldraining lymph nodes are populated with immune cells specific to thecontrol, or undesired, cell immunogens, thereby allowing theSCLC-specific immune cells to be “captured” in the node close to thesite of immunization with the SCLC cells as immunogen. Other methods ofimmunization and protocols for eliciting an immune response can beconsidered to be suitable for generating antibodies according to theinvention, see, for example, WO 99/44583.

[0047] The induction of high zone tolerance to an antigen or set ofantigens can be viewed as reshaping the repertoire of the immune system.Immunizing the animal with a lower dose of antigen evokes a stronghumoral response against the minor or weak epitopes due to theirliberation from the shadow created by the dominant epitopes.

[0048] Tolerance is the failure of the immune system to respond to anantigen. Tolerance to self-antigen is an essential feature of the immunesystem that prevents self-inflicted damage to the host's tissues andorgans. When tolerance is lost, the immune system can destroyself-tissues, as happens in autoimmune diseases. Autoimmune disordersare rare and are known to be linked to HLA gene composition. Theprecipitating event that leads to the collapse of self-tolerance isunknown, but is suspected to be associated with infectious agents. Thedeliberate rupture of tolerance is a major challenge and can be achievedfor some antigens using repetitive immunizations in the presence ofextremely powerful adjuvants.

[0049] The production of a monoclonal antibody to a non-dominant, (e.g.,self-antigen), epitope is often desired, but may be hindered, becauseknown immunodominant epitopes overwhelm the immune response. A techniquesuch as contrasting or differential immunization with antigen allowsfocused antibody responses targeting the desired epitope by changing thehierarchy of the antigen dominance. By tolerizing the immunized hostagainst the dominant epitopes, the minor or weaker antigens are“liberated” from the overbearing shadow of the dominant epitopes and,hence, are briskly reacted against by the cells of the immune system.One way to produce monoclonal antibodies directed to an array ofnon-dominant epitopes is by essentially tolerizing immunized animals tothe immunodominant epitopes, followed by immunization with thenon-dominant epitopes, e.g., those epitopes present on the surfaceantigens of SCLC cells.

[0050] The differential or contrasting immunization strategy is alsoable to detect antigens that are of broad chemical diversity. Thisunique capability can be applied to the discovery of antigens ofimportance in areas where gene discovery and gene product technology areunable to function due to the limitation of these technologies for usein the discovery of protein markers and products. Because this type ofimmunization is suitable for generating monoclonal antibodies against abroad range of chemically diverse antigens, the technology is applicablefor the development of protein markers and therapeutic productspertaining to cancers, tumors (both benign and malignant), infectiousagents, stem cell transplantation, neurodegenerative diseases,cardiovascular diseases, autoimmune diseases, allergic diseases andinflammatory diseases.

[0051] For example, the contrasting immunization protocol is used totolerize mice to a pool of human neuroendocrine cells, e.g., theneuroblastoma cell lines SK-N-AS, Be (2)-M17, and MC-IXC (Example 1),which are closely related by lineage to SCLC cells. The tolerized miceare then immunized with a pool of three human SCLC cell lines. Serumfrom mice immunized with the differential protocol contain antibodiesthat are specific to human SCLC cells, while serum from mice immunizedusing conventional methods are devoid of such SCLC cancer-specificantibodies.

[0052] The present invention provides the discovery of new monoclonalantibodies (MoAbs) directed against cancer-specific antigen(s) expressedon human SCLC cells. Preferred is the use of the contrasting ordifferential immunization technique to elicit monoclonal antibodieshaving specificities toward surface antigen or marker epitopes of SCLCcancer or tumor cells. The monoclonal antibodies were characterized andfound to bind specifically to antigens on the surface of SCLC cells. Inparticular, three representative, exemplary monoclonal antibodies wereisolated, characterized and found to react specifically with a singlechain SCLC cell surface glycoprotein with a MW of about 200 KDa (asdetermined by SDS-PAGE under reducing conditions) present on SCLC cells(FIGS. 2, 3 and 8B). These MoAbs were given the designations 51.2, 37.14and 109.12 (ATCC Designation Nos. PTA-2360, PTA-2358 and PTA-2357,respectively). MoAb 109.12 was found not to react with developmentallyrelated neuroendocrine cells, i.e., neuroblastoma cell lines, orunrelated human multiple myeloma (MM) cancer cells. MoAb 51.2 reactedonly minimally with neuroendocrine cells and not at all with MM cells.

[0053] The representative isolated monoclonal antibody, 37.14, was alsofound to react specifically with a cell surface glycoprotein having MWof between about 35-50 kDa as determined by SDS-PAGE under reducingconditions. This antibody was deposited with the ATCC under ATCCDesignation No. PTA-2358. The 35-50 kDa antigen appeared to be comprisedof two chains or fragments, closely related in mass. The 37.14 antibodydid not recognize any similar antigen on either neuroendocrine cells orunrelated MM cells.

[0054] Yet another representative isolated monoclonal antibody, MoAb26.1 (deposited with the ATCC under ATCC Designation No. PTA-2359), wasfound to recognize a SCLC-specific cell surface molecule as assayedunder conditions for FACS analysis. However, this antibody did notappear to react with SCLC-membranes in Western Blot assays performedunder reducing conditions. Such a result suggests that the epitoperecognized by MoAb 26.1 is a conformational epitope that is destroyed bylinearization under the conditions for Western Blotting involving theuse of SDS.

[0055] Another embodiment of the present invention relates to monoclonalantibodies, and binding fragments or portions thereof, which recognizethe foregoing SCLC cell surface glycoproteins. Thus, the presentinvention encompasses the deposited monoclonal antibodies, as well asantibodies, preferably monoclonal antibodies, and their bindingfragments, having specificity for the above-described antigens presenton SCLC cells. Nonlimiting examples of antibody fragments or antigenbindable fragments that bind to epitopes on the SCLC antigens includethe following: Fab fragments, F(ab)₂ fragments, Fab′ fragments,fragments produced by F(ab) expression libraries, F(ab′)₂ fragments, Fdfragments, Fd′ fragments and Fv fragments. The antibodies may be human,or from animals other than humans, preferably mammals, such as rat,mouse, guinea pig, rabbit, goat, sheep, and pig. Preferred are mousemonoclonal antibodies and antigen-binding fragments or portions thereof.In addition, chimeric antibodies and hybrid antibodies are embraced bythe present invention.

[0056] In accordance with the present invention, the monoclonalantibodies and binding fragments thereof may be characterized as thosewhich are 1) produced from the hybridoma cell lines deposited at theAmerican Type Culture Collection, 10801 University Boulevard, Manassas,Va., 20110-2209 under ATCC Accession Nos. PTA-2357, PTA-2358, PTA-2359and PTA-2360; 2) antibodies that are capable of binding to the sameantigenic determinant as does the monoclonal antibody produced by thehybridoma cell lines deposited at the American Type Culture Collectionunder ATCC Accession Nos. PTA-2357, PTA-2358, PTA-2359 and PTA-2360; 3)binding fragments of the monoclonal antibodies produced by the hybridomacell lines deposited at the American Type Culture Collection under ATCCAccession Nos. PTA-2357, PTA-2358, PTA-2359 and PTA-2360; or 4) bindingfragments of a monoclonal antibody capable of binding to the sameantigenic determinant as does the monoclonal antibody produced by thehybridoma cell lines deposited at the American Type Culture Collectionunder ATCC Accession Nos. PTA-2357, PTA-2358, PTA-2359 and PTA-2360. Theimmunoglobulin isotypes of the deposited monoclonal antibodies of thepresent invention are presented in Table 1 as follows: TABLE I MoAbIsotype (H, L chain) 21.7 IgG1, kappa 26.1 IgG1, kappa 37.14 IgG2a,kappa 51.2 IgG2a, kappa 109.12 IgG1, kappa

[0057] According to the present invention, the monoclonal antibodiesrecognize specific glycoprotein surface antigens expressed by andpresent on SCLC cells. The glycoprotein surface antigen recognized by asubset of the monoclonal antibodies as represented by MoAb 37.14, MoAb51.2 and MoAb 109.12, ATCC Accession Nos. PTA-2358, PTA-2360 andPTA-2357, respectively, is a single polypeptide having a molecularweight of about 200 KDa as determined by SDS-PAGE under reducingconditions. The glycoprotein surface antigen, or epitope, recognized byanother subset of the monoclonal antibodies represented by MoAb 37.14,ATCC Accession No. PTA-2358, has a molecular weight of about 35-50 KDaas determined by SDS-PAGE under reducing conditions. SCLC-specificantigens recognized by others of the monoclonal antibodies according tothe present invention contain conformational epitopes, whose recognitionby the monoclonal antibodies as represented by MoAb 26.1 is dependent onthe conformational nature of the antigen being intact and not denatured,degraded, linearized, or otherwise adversely affected.

[0058] The present invention further provides hybridoma cell lines thatproduce a monoclonal antibodies that specifically bind to antigens onthe surface of SCLC cells as described and characterized herein. Methodsfor preparing hybridoma cell lines are well known and practiced in theart. Accordingly, any technique or protocol that results in theproduction of homogeneous populations of antibody molecules to aspecific antigen, preferably monospecific antibody molecules, e.g.,monoclonal antibodies, by continuous cell lines in culture may be used.Such techniques include, but are not limited to, the hybridoma techniquedeveloped by Kohler and Milstein (1975, Nature, 256:495-497), the triomatechnique, the human B-cell hybridoma technique (Kozbor et al., 1983,Immunology Today, 4:72; Cote et al., 1983, Proc. Nat'l Acad. Sci. USA,80:2026-2030), as well as the Epstein Barr Virus (EBV)-hybridomatechnique to produce human monoclonal antibodies (Cole et al., 1985, In:Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp.77-96). Such antibodies may be of any immunoglobulin class, includingIgM, IgG, IgE, IgA and IgD, or any subclass thereof. Hybridoma cells maybe cultured in vivo or in vitro according to established methods.

[0059] In a further aspect, monoclonal antibodies can be produced ingerm-free animals utilizing the technology described in InternationalPatent Application No. WO 98/02545. Also suitable for use in the presentinvention are hybrid antibodies, chimeric antibodies and humanizedantibodies (e.g., U.S. Pat. No. 5,585,089 to Queen et al.). Antibodies,such as hybrid or chimeric antibodies having human components, orhumanized antibodies, are preferable for use in therapies of humandiseases or disorders, because the human or humanized antibodies aremuch less likely than xenogenic antibodies to induce an immune response,particularly an allergic response, when introduced into a human host.

[0060] In addition, techniques developed for the production of chimericantibodies (Morrison et al., 1984, Proc. Natl. Acad. Sci. USA,81:6851-6855; Neuberger et al., 1984, Nature, 312:604-608, Takeda etal., 1985, Nature, 314:452-454) by splicing the genes from a mouseantibody molecule of appropriate antigen specificity together with genesfrom a human antibody molecule of appropriate biological activity aresuitable for use in the present invention.

[0061] Further, according to the present invention, the techniquesdescribed for the production of single chain antibodies (e.g., U.S. Pat.Nos. 5,476,786 and 5,132,405 to Huston; Huston et al., 1988, Proc. Natl.Acad. Sci. USA, 85:5879-5883; U.S. Pat. No. 4,946,778 to R. C. Ladner etal.; Bird, 1988, Science, 242:423-426 and Ward et al., 1989, Nature,334:544-546) can be adapted to produce SCLC-surface antigen specificsingle chain antibodies. Single chain antibodies are formed by linkingthe heavy and light immunoglobulin chain fragments of the Fv region viaan amino acid bridge, resulting in a single chain polypeptide. Univalentantibodies are also embraced by the present invention. In addition,techniques for the construction of Fab expression libraries (Huse etal., 1989, Science, 246:1275-1281) are suitable for use in thisinvention to allow the rapid and easy identification of monoclonalantibody Fab fragments, or derivatives or analogs, having the desiredspecificity.

[0062] Antibody fragments containing the idiotype of the specificanti-SCLC surface antigen antibodies, and which “mimic” the SCLCspecific antigens, can be produced by known techniques (Greenspan andBona, 1993, FASEB J., 7(5):437-444 and Nissinoff, 1991, J. Immunol.,147(8):2429-2438). For example, such fragments include, withoutlimitation, the F(ab′)₂ fragment, which can be produced by pepsindigestion of the intact antibody molecule; the Fab′ fragment, which canbe produced by reducing the disulfide bridges of the F(ab′)₂ fragments,and the Fab fragments, which can be generated by treating the intactantibody molecule with the enzyme papain and a reducing agent.

[0063] It is to be understood that the contrasting immunization anddifferential antigen immunization procedures described herein arenonlimiting examples of ways in which the desired antibodies can beobtained for their numerous uses. Accordingly, it is also envisionedthat anti-SCLC antibodies can be elicited in an animal host byimmunization with SCLC cells or cell-derived immunogenic components, orcan be formed by in vitro immunization (sensitization) of immune cells.The antibodies can also be produced in recombinant systems in which theappropriate cell lines are transformed, transfected, infected ortransduced with appropriate antibody-encoding DNA. Alternatively, theantibodies can be constructed by biochemical reconstitution of purifiedheavy and light chains. Using the aforementioned types of antibodies,for example, cells displaying the specifically recognized surfaceglycoprotein antigens, or the antigens themselves, or an immunogenicfragment or portion thereof, can be detected in a test sample bychromatography on antibody-conjugated solid-phase matrices or supports(see E. Harlow and D. Lane, 1999, Using Antibodies: A Laboratory Manual,Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.), or byimmunoassay. Preferred are antibodies that specifically recognize andbind to surface antigens expressed by SCLC cells; more preferred are theSCLC specific surface glycoprotein antigens described herein.

[0064] The antibodies can be employed to prepare anti-SCLC antigenantibody affinity columns. For example, gel supports or beads can beactivated with various chemical compounds, e.g., cyanogen bromide,N-hydroxysuccinimide esters, and antibodies can be bound thereto. Moreparticularly and by way of example, anti-SCLC antibodies can be added toAffigel-10 (Biorad), a gel support which is activated withN-hydroxysuccinimide esters, such that the antibodies form covalentlinkages with the agarose gel bead support. The antibodies are thencoupled to the gel via amide bonds with a spacer arm. The remainingactivated esters are then quenched with ethanolamine HCl, IM, pH 8. Thecolumn is washed with water, followed by 0.23M glycine HCl, pH 2.6, toremove any non-conjugated antibody or extraneous protein. The column isthen equilibrated in phosphate buffered saline (PBS), (pH 7.3) withappropriate detergent, and the sample materials, i.e., cell culturesupernatants or cell extracts, for example, containing SCLC surfaceantigen (e.g., prepared using appropriate membrane solubilizingsurfactants) are slowly passed over the column. The column is washedwith PBS/surfactant until the optical density falls to background. Theprotein is then eluted from the column with 0.23 M glycine-HCl, pH2.6/surfactant. The purified SCLC-derived glycoprotein is then dialyzedagainst PBS/surfactant.

[0065] In another embodiment, the present invention embraces one or moreisolated surface antigens of SCLC, as described herein and as recognizedand bound by the monoclonal antibodies according to this invention. Inparticular, the invention embraces the approximately 200 KDa singlechain glycoprotein antigen of SCLC cells recognized by the 37.14, 109.12and 51.2 monoclonal antibodies having ATCC Accession No. PTA-2358, ATCCAccession No. PTA-2357 and ATCC Accession No. PTA-2360, respectively, aswell as the approximately 35-50 KDa single chain glycoprotein antigen orepitopic determinant on SCLC cells as recognized by the 37.14 monoclonalantibody having ATCC Accession No. PTA-2358. Further embraced by thepresent invention are cell surface glycoproteins comprising an epitoperecognized by the antibodies of this invention, e.g., the 26.1 MoAbhaving ATCC Accession No. PTA-2359. The cell surface glycoproteinscomprising the recognized conformational epitope are not present onnormal cells, or on cells that are developmentally unrelated to SCLC.

[0066] Another aspect of the present invention relates to therapeuticmethods for the treatment of individuals afflicted with SCLC andneuroendocrine diseases involving cells displaying the SCLC-specificsurface glycoprotein(s), particularly, those having a molecular weightof about 200 KDa or of about 35-50 KDa, as determined by SDS-PAGE underreducing conditions and recognized by the antibodies according to thisinvention. Other dysproliferative diseases in which the glycoproteinantigens described herein are present on the cell surface are alsotreatable using the antibodies and methods according to this invention.

[0067] The therapeutic methods encompassed by the present inventioninvolve primary tumors or cancers, as well as metastases. As an example,a method for inhibiting or killing SCLC cells comprises administering toa patient one or more of the monoclonal antibodies having specificityfor SCLC cells, or a binding fragment thereof, as described above, underconditions sufficient for the binding of the monoclonal antibody, orbinding fragment, to tumor or cancer cells in the patient. The bindingof antibodies, or their binding fragments, to the tumor cells or cancercells induces the inhibiting or killing of the cells by the patient'simmune cells. The above described method employs the antibodies or theirbinding fragments without modification, relying on the binding of theantibodies to the surface of the SCLC cells in situ to stimulate andinduce an immune response and attack by autologous immune cells thereon.However, such antibody-mediated treatment or therapy may also beaccompanied by other treatments that are directed to tumor or cancercells, for example, radiation, chemotherapy, and the like, as well as byadjunctive therapies to enhance the immune system's attack on theopsonized cancer or tumor cells following the above-describedtreatment/therapy procedure(s).

[0068] More specifically, a growth factor, lymphokine, or cytokine maybe co-administered with one or more of the anti-SCLC monoclonalantibodies, for example, erythropoietin and/or GM-CSF, to stimulatewhite blood cells and support the immunocompetence status of thepatient. In addition, chimeric or fusion antibodies, or otherrecombinant antibodies of the present invention may be used in therapiesand treatment. For example, a fusion protein molecule comprising atleast the antigen-binding region of an antibody of the invention joinedto at least a functionally active or bioactive portion of a secondprotein having anti-tumor or cancer effects, e.g., a lymphokine oroncostatin, may be used to treat SCLC or tumors, particularly, in vivo.Moreover, a chimeric antibody can be prepared, wherein the antigenbinding portion or site is joined to a human Fc molecule of animmunoglobulin, e.g., IgG1, to promote antibody-dependent mediatedcytotoxicity or complement-mediated cytotoxicity. Recombinant techniquesand protocols as known and practiced in the art (e.g., U.S. Pat. No.4,474,893 to C. L. Reading, issued Oct. 2, 1984) may be used toconstruct bispecific or bifunctional chimeric antibodies wherein one ofthe binding specificities is that of the antibody according to thepresent invention.

[0069] In another of its aspects, the present invention comprisestherapeutic methods utilizing the described monoclonal antibodies, orbinding fragments thereof, to which a cytotoxic agent has been bound,affixed or coupled. The binding of the cytotoxic antibodies, or bindingfragments thereof, to the SCLC tumor or cancer cells inhibits the growthof the cells and optimally kills the cells. Nonlimiting examples ofsuitable cytotoxic agents include chemotherapeutic compounds, a drug(e.g., Garnett and Baldwin, 1986, Cancer Res., 46:2407-24112), aprodrug, enzymes, a photoactivated toxin, or a radioactive agent.Cytotoxic agents include, but are not limited to, ricin A chain, abrin Achain, modeccin A chain, gelonin, melphalan, bleomycin, adriamycin,daunomycin, or pokeweed antiviral proteins (PAP, PAPII, or PAP-S).

[0070] The skilled practitioner will realize that there are numerousradionuclides and chemocytotoxic agents that can be coupled totumor-specific antibodies by well-known techniques and delivered to asite to specifically destroy tumor cells and tissue. (See, for example,U.S. Pat. No. 4,542,225 to W. A. Blattler et al., issued Sep. 17, 1985;and Pastan et al., 1986, Cell, 47:641-648). Nonlimiting examples ofphotoactivated toxins include dihydropyridine- and omega-conotoxin(Schmidt et al., 1991, J. Biol. Chem., 266(27):18025-18033). Nonlimitingexamples of imaging and cytotoxic reagents that are suitable for useinclude ¹²⁵I, ¹²³I, ¹¹¹In (e.g., Sumerdon et al., 1990, Nucl. Med.Biol., 17:247-254), ^(99m)Tc, ³²P, ³H and ¹⁴C; fluorescent labels suchas fluorescein and rhodamine; chemiluminescent labels such as luciferin,and paramagnetic ions for use in magnetic resonance imaging (Lauffer etal., 1991, Magnetic Resonance in Medicine, 22:339-342). Antibodies canbe labeled with such reagents using protocols and techniques known andpracticed in the art. See, for example, Wenzel and Meares,Radioimmunoimaging and Radioimmunotherapy, Elsevier, New York, 1983;Colcer et al., 1986, Meth. Enzymol., 121:802-816; and MonoclonalAntibodies for Cancer Detection and Therapy, Eds. Baldwin et al.,Academic Press, 1985, pp. 303-316, for techniques relating to theradiolabeling of antibodies. Yttrium-90 labeled monoclonal antibodieshave been described for maximizing the dose delivered to the tumor orcancer cells and/or tissue, while limiting toxicity to normal tissues(e.g., Goodwin and Meares, 1997, Cancer Supplement, 80:2675-2680). Othercytotoxic radionuclides including, but not limited to, Copper-67 (⁶⁷Cu),Iodine-131 (¹³¹I) and Rhenium-186 can also be used for labelingmonoclonal antibodies directed against SCLC surface antigens.

[0071] The detectable/detecting label used is selected according to theimaging modality to be used. For example, radioactive labels, such asIndium-111 (¹¹¹In), Technetium-99m (^(99m)Tc), or Iodine 131 (¹³¹I), canbe used for planar scans or for single photon emission computedtomography (SPECT). Also, positron-emitting labels such as Fluorine-19can be used in positron emission tomography (PET). Paramagnetic ions,such as Gadlinium(III) or Manganese(II) can be used in magneticresonance imaging (MRI). The monoclonal antibodies can also be labeledwith radio-opaque labels for the visualization of SCLC cells afterinjection, for example, by X-ray, CATscan, or MRI. In particular, forSCLC disease, localization of the label within the lung, or external tothe lung, permits the determination of the spread of the disease. Theamount of label that is present and detectable within the lung, forexample, allows the determination of the presence or absence of canceror tumor in the lung.

[0072] Other covalent and non-covalent modifications of the monoclonalantibodies, or their binding fragments, as described herein are furtherencompassed for use in the present invention. Such modifications aremeant to include agents which are co-administered with, or areadministered subsequent to, the administration of the antibody(ies), orfragments thereof, to induce or stimulate growth inhibition or killingof the cells to which the antibody(ies) or fragments bind. For example,immunotoxins conjugated to monoclonal antibodies have been found to beefficacious in animal models. The conjugation of MoAbs withribosome-inactivating proteins (e.g., ricin A-chain, ricinus agglutinin,or viscumin) or photoinactivating agents has been described (see, e.g.,D. B. Papkovskii et al., 1990, Biomed. Sci., 1(4):401-406). In addition,pokeweed antiviral protein (PAP) has the ability to disruptanti-apoptotic complexes or inhibit protein synthesis within the targetcell, ultimately resulting in the death of the cell. Further, a numberof small molecules that inhibit tyrosine kinases can be specificallytargeted to cancer cells as growth factor conjugates and which can beadministered with the monoclonal antibodies, or fragments thereof,according to the present invention.

[0073] In a related embodiment of the present invention, the monoclonalantibodies according to this invention can be used for immunotherapy,either unlabeled or labeled with a therapeutic agent. These therapeuticagents can be coupled either directly or indirectly to the describedmonoclonal antibodies, using techniques routinely practiced in the art.One example of indirect coupling is by the use of a spacer moiety.Spacer moieties, in turn, can be either insoluble or soluble (Dieher etal., 1986, Science, 231:148) and can be selected to enable drug releasefrom the monoclonal antibody molecule at the target site. Examples oftherapeutic agents which can be coupled to the monoclonal antibodies ofthe invention for anti-cancer immunotherapy are drugs, radioisotopes,lectins, and toxins.

[0074] The drugs with which can be conjugated to the monoclonalantibodies of the present invention include non-proteinaceous as well asproteinaceous compounds. The term “non-proteinaceous drugs” encompassescompounds which are classically referred to as drugs, for example,mitomycin C, daunorubicin, and vinblastine. The proteinaceous drugs withwhich the monoclonal antibodies of the invention can be labeled includeimmunomodulators and other biological response modifiers.

[0075] The term “biological response modifiers” is meant to encompasssubstances that are involved in modifying the immune response in suchmanner as to enhance the destruction of the antigen-bearing tumor forwhich the monoclonal antibodies of the invention is specific. Examplesof immune response modifiers include such compounds as lymphokines.Lymphokines include tumor necrosis factor, interleukins, e.g., IL1through IL15, lymphotoxin, macrophage activating factor (MAF), migrationinhibition factor (MIF), colony stimulating factor (CSF), andinterferon. Interferons with which the monoclonal antibodies of theinvention can be labeled include alpha-interferon, beta-interferon andgamma-interferon and their subtypes.

[0076] In using radioisotopically conjugated monoclonal antibodies ofthe invention for immunotherapy, certain isotopes may be more preferablethan others depending on such factors as leukocyte distribution as wellas isotope stability and emission. If desired, the tumor celldistribution can be evaluated by the in vivo diagnostic techniquesdescribed above. Depending on the malignancy, some emitters may bepreferable to others. In general, alpha and beta particle-emittingradioisotopes are preferred in immunotherapy. For example, if an animalhas solid tumor foci, as in a carcinoma, a high energy beta emittercapable of penetrating several millimeters of tissue, such as ⁹⁰Y, maybe preferable. On the other hand, if the malignancy consists of simpletarget cells, as in the case of leukemia, a shorter range, high energyalpha emitter, such as ²¹²Bi, may be preferable. Examples ofradioisotopes which can be bound to the monoclonal antibodies of theinvention for therapeutic purposes are ¹²⁵I, ¹³¹I, ⁹⁰Y, ⁶⁷Cu, ²¹²Bi,²¹¹At, ²¹²Pb, ⁴⁷Sc, ¹⁰⁹Pd, and ¹⁸⁸Re.

[0077] Lectins are proteins, usually isolated from plant material, whichbind to specific sugar moieties. Many lectins are also able toagglutinate cells and stimulate lymphocytes. Ricin is a toxic lectinthat has been used immunotherapeutically. This is preferablyaccomplished by binding the alpha-peptide chain of ricin, which isresponsible for toxicity, to the antibody molecule to enable sitespecific delivery of the toxic effect.

[0078] Toxins are poisonous substances produced by plants, animals, ormicroorganisms that, in sufficient dose, are often lethal. Diphtheriatoxin (DT), a substance produced by Corynebacterium diphtheria, can beused therapeutically. DT consists of an alpha and beta subunit whichunder proper conditions can be separated. The toxic alpha component canbe bound to an antibody and used for site specific delivery to a cellbearing an antigen for which the monoclonal antibodies of the inventionare specific. Other therapeutic agents which can be coupled to themonoclonal antibodies of the invention are known, or can be easilyascertained, by those of ordinary skill in the art.

[0079] The labeled or unlabeled monoclonal antibodies of the presentinvention can also be used in combination with therapeutic agents suchas those described above. Especially preferred are therapeuticcombinations comprising the monoclonal antibody of the invention andimmunomodulators and other biological response modifiers. Thus, forexample, the monoclonal antibodies of the invention can be used incombination with alpha-interferon. This treatment modality enhancesmonoclonal antibody targeting of carcinomas by increasing the expressionof monoclonal antibody reactive antigen by the carcinoma cells (Greineret al., 1987, Science, 235:895). Alternatively, the monoclonalantibodies of this invention may be used, for example, in combinationwith gamma-interferon to activate and increase the expression of Fcreceptors by effector cells, which, in turn, results in an enhancedbinding of the monoclonal antibody to the effector cell and killing oftarget tumor cells. Those of skill in the art will be able to selectfrom the various biological response modifiers to create a desiredeffector function which enhances the efficacy of the monoclonalantibodies of the invention.

[0080] When the monoclonal antibodies of the present invention are usedin combination with various therapeutic agents, such as those describedherein, the administration of the monoclonal antibody and thetherapeutic agent usually occurs substantially contemporaneously. Theterm “substantially contemporaneously” means hat the monoclonal antibodyand the therapeutic agent are administered reasonably close togetherwith respect to time. Usually, it is preferred to administer thetherapeutic agent before the monoclonal antibody. For example, thetherapeutic agent can be administered 1 to 6 days before the monoclonalantibody. The administration of the therapeutic agent can be daily, orat any other interval, depending upon such factors, for example, as thenature of the tumor, the condition of the patient and the half-life ofthe agent.

[0081] Using the monoclonal antibodies of the present invention, it ispossible to design therapies combining all of the characteristicsdescribed herein. In a given situation, it may be desirable toadminister a therapeutic agent, or agents, prior to the administrationof the monoclonal antibodies of the invention, in combination witheffector cells and the same, or different, therapeutic agent or agents.For example, it may be desirable to treat patients with malignantdisease by first administering gamma-interferon and interleukin-2 dailyfor 3 to 5 days, and on day 5 administer the monoclonal antibody of theinvention in combination with effector cells, as well asgamma-interferon, and interleukin-2.

[0082] It is also possible to utilize liposomes with the monoclonalantibodies of the present invention in their membranes to specificallydeliver the liposome to the area of the tumor expressing SCLC-specificantigens. These liposomes can be produced such that they contain, inaddition to monoclonal antibody, immunotherapeutic agents, such as thosedescribed above, which would then be released at the tumor site (e.g.,Wolff et al., 1984, Biochem. et Biophys. Acta, 802:259).

[0083] The dosage ranges for the administration of the monoclonalantibodies of the invention are those large enough to produce thedesired effect in which the symptoms of the malignant disease areameliorated. The dosage should not be so large as to cause adverse sideeffects, such as unwanted cross-reactions, anaphylactic reactions, andthe like. Generally, the dosage will vary with the age, condition, sexand extent of disease of the patient and can be determined by one ofskill in the art. The dosage can be adjusted by the individual physicianin the event of any complication. Dosage can vary from about 0.1 mg/kgto about 2000 mg/kg, preferably about 0.1 mg/kg to about 500 mg/kg, inone or more dose administrations daily, for one or several days.

[0084] Generally, when the monoclonal antibodies of the presentinvention are administered conjugated with therapeutic agents, lowerdosages, comparable to those used for in vivo immunodiagnostic imaging,can be used. The monoclonal antibodies of the invention can beadministered parenterally by injection or by gradual perfusion overtime. The monoclonal antibodies of the invention can be administeredintravenously, intraperitoneally, intramuscularly, subcutaneously,intracavity, or transdermally, alone or in combination with effectorcells. Preparations for parenteral administration include sterileaqueous or non-aqueous solutions, suspensions, and emulsions. Examplesof non-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's, or fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives may also be present such as,for example, antimicrobials, anti-oxidants, chelating agents, and inertgases and the like.

[0085] As mentioned above, anti-idiotypic monoclonal antibodies to theantibodies according to the present invention may be used in therapiesand treatments in active tumor immunization and tumor therapy (See, S.M. Larson et al., 1986, “Therapeutic applications of radiolabeledantibodies: Current situation and prospects”, Int. J. Rad. Appl.Instrum., B).

[0086] The monoclonal antibodies, or binding fragments thereof,according to the present invention, may be used to quantitatively orqualitatively detect the presence of the SCLC-specific antigens asdescribed on tumor or cancer cells. This can be achieved, for example,by immunofluorescence techniques employing a fluorescently labeledantibody, coupled with light microscopic, flow cytometric, orfluorometric detection. In addition, the antibodies, or bindingfragments thereof, according to the present invention may additionallybe employed histologically, as in immunofluorescence, immunoelectronmicroscopy, or non-immuno assays, for the in situ detection ofSCLC-specific antigen on cells, such as for use in monitoring,diagnosing, or detection assays.

[0087] In situ detection may be accomplished by removing a histologicalspecimen from a patient, and applying thereto a labeled antibodyaccording to this invention. The antibody, or antigen-binding fragmentthereof, is preferably applied by overlaying the labeled antibody orfragment onto the biological sample. Through the use of such aprocedure, it is possible to determine not only the presence of the SCLCantigen, or conserved variants, or peptide fragments, but also itsdistribution in the examined tissue. Those having skill in the art willreadily recognize that any of a wide variety of histological methods,e.g., staining procedures, can be modified in order to achieve such insitu detection.

[0088] Immunoassay and non-immuno assays for SCLC antigen, or conservedvariants, or peptide fragments thereof, typically comprise incubating asample, such as a biological fluid, tissue extract, freshly harvestedcells, or lysates of cells that have been incubated in cell culture, inthe presence of a detectably-labeled antibody that recognizes the SCLCantigen, conserved variants, or peptide fragments thereof, such as theSCLC-specific monoclonal antibodies, or binding fragments thereof, ofthe present invention. Thereafter, the bound antibody, or bindingfragment thereof, is detected by a number of techniques well known inthe art.

[0089] The biological sample may be brought into contact with, andimmobilized onto, a solid phase support or carrier, such asnitrocellulose, or other solid support or matrix, which is capable ofimmobilizing cells, cell particles, membranes, or soluble proteins. Thesupport may then be washed with suitable buffers, followed by treatmentwith the detectably-labeled anti-SCLC antibody. The solid phase supportmay then be washed with buffer a second time to remove unbound antibody.The amount of bound label on the solid support may then be detected byconventional means. Accordingly, in another embodiment of the presentinvention, compositions are provided comprising the monoclonalantibodies, or binding fragments thereof, bound to a solid phasesupport, such as described herein.

[0090] By solid phase support or carrier or matrix is meant any supportcapable of binding an antigen or an antibody. Well-known supports orcarriers include glass, plastic, nylon wool, polystyrene, polyethylene,polypropylene, dextran, nylon, amylases, films, resins, natural andmodified celluloses, polyacrylamides, agarose, alumina gels, gabbros,and magnetite. The nature of the carrier can be either soluble to someextent, or insoluble for the purposes of the present invention. Thesupport material may have virtually any possible structuralconfiguration as long as the coupled molecule is capable of binding toan antigen or antibody. Thus, the support configuration may bespherical, as in a bead, cylindrical, as in the inside surface of a testtube, or the external surface of a rod. Alternatively, the surface maybe flat, such as a sheet, film, test strip, stick, and the like. Inaddition, the solid support is preferably inert to the reactionconditions for binding and may have reactive groups, or activatedgroups, in order to attach the monoclonal antibody, a binding fragment,or the binding partner of the antibody. The solid phase support may alsobe useful as a chromatographic support, such as the carbohydratepolymers Sepharose®, Sephadex®, or agarose. Indeed, a large number ofsuch supports for binding antibody or antigen are commercially availableand known to those having skill in the art.

[0091] The binding activity for a given anti-SCLC antibody may bedetermined by well-known methods. Those skilled in the art will be ableto determine operative and optimal assay conditions for eachdetermination by employing routine experimentation.

[0092] With respect to the anti-SCLC antibodies, numerous ways todetectably label such protein molecules are known and practiced in theart. For example, one way the antibodies can be detectably labeled is bylinking the antibody to an enzyme, e.g., for use in an enzymeimmunoassay (EIA), (A. Voller et al., 1978, “The Enzyme LinkedImmunosorbent Assay (ELISA)”, Diagnostic Horizons, 2:1-7;,Microbiological Associates Quarterly Publication, Walkersville, Md.; A.Voller et al., 1978, J. Clin. Pathol., 31:507-520; J. E. Butler et al.,1981, Meths. Enzymol., 73:482-523; Enzyme Immunoassay, 1980, (Ed.) E.Maggio, CRC Press, Boca Raton, Fla.; Enzyme Immunoassay, 1981, (Eds.) E.Ishikawa et al., Kgaku Shoin, Tokyo, Japan). The enzyme that is bound tothe antibody reacts with an appropriate substrate, preferably achromogenic substrate, so as to produce a chemical moiety which can bedetected, for example, by spectrophotometric, fluorometric, or by visualdetection means. Nonlimiting examples of enzymes which can be used todetectably label the antibodies include malate dehydrogenase,staphylococcal nuclease, delta-5-steroid isomerase, yeast alcoholdehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphateisomerase, horseradish peroxidase, alkaline phosphatase, ribonuclease,urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase andacetylcholinesterase. The detection can be accomplished by calorimetricmethods, which employ a chromogenic substrate for the enzyme, or byvisual comparison of the extent of enzymatic reaction of a substratecompared with similarly prepared standards or controls.

[0093] A variety of other immunoassays may also be used for detection.For example, by labeling the antibodies, or binding fragments thereof,with a radioisotope, a radioimmunoassay (RIA) can be used to detectSCLC-specific antigens (e.g., B. Weintraub, “Principles ofRadioimmunoassays”, Seventh Training Course on Radioligand Techniques,The Endocrine Society, March, 1986). The radioactive isotope label canbe detected by using a gamma counter or a scintillation counter or byradiography.

[0094] The antibodies, or their antigen-binding fragments can also belabeled using a fluorescent compound. When the fluorescently labeledantibody is exposed to light of the proper wavelength, its presence canthen be detected due to fluorescence. Among the most commonly usedfluorescent labeling compounds are, without limitation, fluoresceinisothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin,o-phthaldehyde and fluorescamine. Detectably labeledfluorescence-emitting metals, such as ¹⁵²Eu, or others of the lanthanideseries, can be used to label the antibodies, or their binding fragments,for subsequent detection. The metals can be coupled to the antibodiesvia such metal chelating groups as diethylenetriaminepentacetic acid(DTPA), or ethylenediaminetetraacetic acid (EDTA).

[0095] The antibodies can also be detectably labeled by coupling them toa chemiluminescent compound. The presence of the chemiluminescent-taggedantibody is then determined by detecting the presence of luminescencethat develops during the course of a chemical reaction. Examples ofparticularly useful chemiluminescent labeling compounds include, withoutlimitation, luminol, isoluminol, theromatic acridinium ester, imidazole,acridinium salt and oxalate ester. Similarly, a bioluminescent compoundmay be used to label the antibodies of the present invention.Bioluminescence is a type of chemiluminescence found in biologicalsystems in which a catalytic protein increases the efficiency of thechemiluminescent reaction. The presence of a bioluminescent protein isdetermined by detecting the presence of luminescence. Usefulbioluminescent labeling compounds include luciferin, luciferase andaequorin.

[0096] Another embodiment of the present invention provides diagnostics,diagnostic methods and imaging methods for SCLC cancers and tumors usingthe monoclonal antibodies and binding fragments thereof as described bythe present invention. The diagnostic uses of the antibodies accordingto the present invention embrace primary tumors and cancers, as well asmetastases. Other cancers and tumors bearing the surface antigensdiscovered on SCLC cells, bindable by the monoclonal antibodies, orbinding fragments thereof, of this invention, and described herein arealso amenable to these diagnostic and imaging procedures.

[0097] A diagnostic method according to the invention comprisesadministering, introducing, or infusing the monoclonal antibodies ortheir binding fragments as described herein, with or without conjugationto a detectable moiety, such as a radioisotope. After administration orinfusion, the antibody or binding fragment binds to the tumor or cancercells, after which the location of the bound antibodies or fragments isdetected. For detectably labeled antibodies or fragments, for example,those labeled with a radioisotope, imaging instrumentation may be usedto identify the location of the agent within the body. For unlabeledantibodies or fragments, a second detectable reagent may beadministered, which locates the bound antibodies or fragments so thatthey can be suitable detected. Similar methods have been employed forother antibodies, and the skilled practitioner will be aware of thevarious methods suitable for imaging the location of detectably boundantibodies or fragments within the body. As a nonlimiting guide, about10-1000 μg, preferably about 50-500 μg, more preferably about 100-300μg, most preferably about 200-300 μg of Protein G-purified MoAb areadministered. For mice, for example, using 200 μg MoAb andintraperitoneal (i.p.) administration, MoAb is injected three times aweek for three weeks. For 300 μg MoAb and intraperitoneal (i.p.)administration, MoAb is injected two times a week for three weeks.Applicable doses for humans include about 100-200 mcg/kg, or 350-700mg/m².

[0098] It is to be further understood that a cocktail of differentmonoclonal antibodies, such as a mixture of the specific monoclonalantibodies described herein, or their binding fragments, may beadministered, if necessary or desired, to alleviate SCLC. Indeed, usinga mixture of monoclonal antibodies, or binding fragments thereof, in acocktail to target several antigens, or different epitopes, on cancercells, is an advantageous approach, particularly to prevent evasion oftumor cells and/or cancer cells due to downregulation of one of theantigens.

[0099] In another embodiment, the present invention assists in thediagnosis of cancers and tumors by the identification and measurement ofshed SCLC cell surface glycoprotein in body fluids, such as blood,serum, plasma, sputum and the like. For those cancers that express thesurface antigens described herein, wherein the antigens are recognizedby and immunoreactive with the monoclonal antibodies and their bindingfragments according to the present invention, the ability to detectantigens that are shed or sloughed off from the cancer or tumor cellsprovides a needed means of early diagnosis, thereby affording theopportunity for early treatment. Early detection is especially importantfor those cancers that are difficult to diagnose in their early stages.Measurement of shed surface glycoprotein in a whole blood sample, forexample, by the use of one or more of the antibodies or their bindingfragments according to this invention provides early detection,diagnosis and immediate intervention and/or treatment for the evasivecancer or tumor. Treatment may comprise antibody-based immunotherapy asdescribed above, in combination with other immunomodulatory agents, ifnecessary or desired.

[0100] Moreover, the level of shed antigen that is detected and measuredin a body fluid sample such as blood provides a means for monitoring thecourse of therapy for the cancer or tumor, including, but not limitedto, surgery, chemotherapy, radiation therapy, the therapeutic methods ofthe present invention, and combinations thereof. By correlating thelevel of SCLC-specific antigen in the body fluid with the severity ofdisease, the level of such antigen can be used to indicate successfulremoval of the primary tumor, cancer, and/or metastases, for example, aswell as to indicate and/or monitor the effectiveness of other therapiesover time. For example, a decrease in the level of the cancer ortumor-specific antigen over time indicates a reduced tumor burden in thepatient. By contrast, no change, or an increase, in the level of antigenover time indicates ineffectiveness of therapy, or the continued growthof the tumor or cancer.

[0101] In a related embodiment, the present invention provides methodsfor diagnosing the presence of SCLC by assaying for changes of levels inthe SCLC cell surface antigens in cells, tissues or body fluids comparedwith the levels in cells, tissues, or body fluids, preferably of thesame type, from normal human controls. A change in levels of antigen inthe patient versus the normal human control is associated with thepresence of SCLC. Without limiting this aspect of the present invention,typically, for a quantitative diagnostic assay, a positive resultindicating that the patient being tested has cancer, is one in whichlevels of the SCLC antigen in or on cells, tissues or body fluid are atleast two times higher, and preferably three to five times higher, orgreater, than the levels of the antigens in or on the same cells,tissues, or body fluid of the normal individual as control. Normalcontrols include a human without cancer and/or non-cancerous samplesfrom the patient.

[0102] Another embodiment of the present invention relates topharmaceutical compositions comprising one or more monoclonalantibodies, or binding fragments thereof, according to the invention,together with a physiologically- and/or pharmaceutically-acceptablecarrier, excipient, or diluent. The antibodies, or binding fragments,specifically recognize an SCLC-epitope on one or more SCLC cell surfaceantigens and bind to the antigens. The SCLC-specific cell surfaceantigens are further characterized as described herein and above.

[0103] More specifically, the present invention is directed topharmaceutical compositions comprising a monoclonal antibody, or bindingfragment thereof, including the monoclonal antibodies produced from thehybridoma cell lines deposited at the American Type Culture Collectionhaving ATCC Accession Nos. PTA-2357, PTA-2358, PTA-2359 and PTA-2360;antibodies that are capable of binding to the same antigenic determinantas do the monoclonal antibodies produced by the hybridoma cell linesdeposited at the American Type Culture Collection and having ATCCAccession Nos. PTA-2357, PTA-2358, PTA-2359 and PTA-2360; bindingfragments of the hybridoma cell lines deposited at the American TypeCulture Collection and having ATCC Accession Nos. PTA-2357, PTA-2358,PTA-2359 and PTA-2360; and binding fragments of monoclonal antibodycapable of binding to the same antigenic determinant as do themonoclonal antibodies produced by the hybridoma cell lines deposited atthe American Type Culture Collection and having ATCC Accession No.PTA-2357, PTA-2358, PTA-2359 and PTA-2360; and apharmaceutically-acceptable carrier or diluent. Antibody fragmentsinclude but are not limited to F(ab′)₂ fragments, F(ab) fragments,fragments produced by an F(ab) expression library, Fv fragments, Fd′fragments, or Fd fragments.

[0104] Preferably, the antibodies or binding fragments thereof aredelivered parenterally, such as by intravenous, subcutaneous, orintraperitoneal administration, e.g., injection. Suitable buffers,carriers, and other components known to the art can be used informulating a composition comprising the antibody or fragments forsuitable shelf-life and compatibility with administration. Thesesubstances may include ancillary agents such as buffering agents andprotein stabilizing agents (e.g., polysaccharides).

[0105] More specifically, therapeutic formulations of the antibodies, orbinding fragments thereof, are prepared for storage by mixing theantibodies or their binding fragments, having the desired degree ofpurity, with optional physiologically acceptable carriers, excipients,or stabilizers (Remington's Pharmaceutical Sciences, 17th edition, (Ed.)A. Osol, Mack Publishing Company, Easton, Pa., 1985), in the form oflyophilized cake or aqueous solutions. Acceptable carriers, excipientsor stabilizers are nontoxic to recipients at the dosages andconcentrations employed, and include buffers such as phosphate, citrate,and other organic acids; antioxidants including ascorbic acid; lowmolecular weight (less than about 10 residues) polypeptides; proteins,such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymerssuch as polyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;salt-forming counterions such as sodium; and/or nonionic surfactantssuch as Tween, Pluronics or polyethylene glycol (PEG).

[0106] The antibodies, or binding fragments thereof, also may beentrapped in microcapsules prepared, for example, by coacervationtechniques or by interfacial polymerization (for example,hydroxymethylcellulose or gelatin-microcapsules andpoly-[methylmethacylate] microcapsules, respectively), in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules), or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences,supra.

[0107] Antibodies or their binding fragments to be used for in vivoadministration must be sterile. This is readily accomplished byfiltration through sterile filtration membranes, prior to or followinglyophilization and reconstitution. The antibodies, or binding fragmentsthereof, ordinarily will be stored in lyophilized form or in solution.

[0108] Therapeutic antibody compositions generally are placed into acontainer having a sterile access port, for example, an intravenoussolution bag or vial having a stopper pierceable by a hypodermicinjection needle. The route of administration of the antibodies, orbinding fragments thereof, in accordance with the present invention, isin accord with known methods, e.g., injection or infusion byintravenous, intraperitoneal, intramuscular, intraarterial,subcutaneous, intralesional routes, by aerosol or intranasal routes, orby sustained release systems as noted below. The antibodies, or bindingfragments thereof, are administered continuously by infusion or by bolusinjection. Suitable examples of sustained-release preparations includesemipermeable matrices of solid hydrophobic polymers containing theprotein, which matrices are in the form of shaped articles, e.g., films,or microcapsules. Examples of sustained-release matrices includepolyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) asdescribed by Langer et al., 1981, J. Biomed. Mater. Res., 15:167-277 andLanger, 1982, Chem. Tech., 12:98-105), or poly(vinylalcohol)],polylactides (U.S. Pat. No. 3,773,919; EP 58,481), copolymers ofL-glutamic acid and gamma ethyl-L-glutamate (Sidman et al., 1983,Biopolymers, 22:547-556), non-degradable ethylene-vinyl acetate (Langeret al., supra), degradable lactic acid-glycolic acid copolymers such asthe LUPRON DEPOT.TM. (injectable microspheres composed of lacticacid-glycolic acid copolymer and leuprolide acetate), andpoly-D-(−)-3-hydroxybutyric acid (EP 133,988).

[0109] While polymers such as ethylene-vinyl acetate and lacticacid-glycolic acid enable release of molecules for over 100 days,certain hydrogels release proteins for shorter time periods. Whenencapsulated antibodies remain in the body for a long time, they maydenature or aggregate as a result of exposure to moisture at 37° C.,resulting in a loss of biological activity and possible changes ineffectiveness. Rational strategies can be devised for antibodystabilization depending on the mechanism involved. For example, if theaggregation mechanism is discovered to be intermolecular S—S bondformation through thio-disulfide interchange, stabilization may beachieved by modifying sulfhydryl residues, lyophilizing from acidicsolutions, controlling moisture content, using appropriate additives,and developing specific polymer matrix compositions.

[0110] Sustained-release antibody compositions also include liposomallyentrapped antibodies, or their binding fragments. Liposomes containingthe antibodies are prepared by known methods, for example, DE 3,218,121;Epstein et al., 1985, Proc. Natl. Acad. Sci. USA, 82:3688-3692; Hwang etal., 1980, Proc. Natl. Acad. Sci. USA, 77:4030-4034; EP 52,322; EP36,676; EP 88,046; EP 143,949; EP 142,641; Japanese patent application83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324.Ordinarily the liposomes are of the small (about 200-800 Angstroms)unilamellar type in which the lipid content is greater than about 30mol. % cholesterol, the selected proportion being adjusted for theoptimal antibody therapy.

[0111] An effective amount of antibody to be employed therapeuticallywill depend, for example, upon the therapeutic and treatment objectives,the route of administration, the age, condition, and body mass of thepatient undergoing treatment or therapy, and auxiliary or adjuvanttherapies being provided to the patient. Accordingly, it will benecessary and routine for the practitioner to titer the dosage andmodify the route of administration, as required, to obtain the optimaltherapeutic effect. A typical daily dosage might range from about 1mg/kg to up to about 100 mg/kg or more, preferably from about 1 to about10 mg/kg/day depending on the above-mentioned factors. Typically, theclinician will administer antibody until a dosage is reached thatachieves the desired effect. The progress of this therapy is easilymonitored by conventional assays.

[0112] Various adjuvants may be used to increase the immunologicalresponse to the SCLC antigen and to elicit specific anti-SCLC antibodiesaccording to the present invention. Depending on the host species to beimmunized, adjuvants may include, but are not limited to, Freund's(complete and incomplete), mineral gels, such as aluminum hydroxide,surface active agents, such as lysolecithin, pluronic polyols,polyanions, peptides, oil emulsions, keyhole limpet hemocyanin,dinitrophenol, and potentially useful human adjuvants such as BCG(bacille Calmette-Guerin) and Corynebacterium parvum.

[0113] The antibodies of the present invention are also useful for invitro diagnostic applications for the detection of human SCLC cells, orneuroendocrine cells, e.g., neuroblastoma, that possess the antigen forwhich the antibodies are specific. As detailed above, in vitrodiagnostic methods include immunohistological or immunohistochemicaldetection of tumor cells (e.g., on human tissue, or on cells dissociatedfrom excised tumor specimens), or serological detection of tumorassociated antigens (e.g., in blood samples or other biological fluids).Immunohistochemical techniques involve staining a biological specimen,such as a tissue specimen, with one or more of the antibodies of theinvention and then detecting the presence on the specimen ofantibody-antigen complexes comprising antibodies bound to the cognateantigen. The formation of such antibody-antigen complexes with thespecimen indicates the presence of SCLC in the tissue.

[0114] Detection of the antibody on the specimen can be accomplishedusing techniques known in the art such as immunoenzymatic techniques,e.g., immunoperoxidase staining technique, or the avidin-biotintechnique, or immunofluorescence techniques (see, e.g., Ciocca et al.,1986, “Immunohistochemical Techniques Using Monoclonal Antibodies”,Meth. Enzymol., 121:562-79 and Introduction to Immunology, Ed. Kimball,(2^(nd) Ed), Macmillan Publishing Company, 1986, pp. 113-117). Serologicdiagnostic techniques involve the detection and quantification oftumor-associated antigens that have been secreted or ‘shed’ into theserum or other biological fluids of patients thought to be sufferingfrom SCLC, as mentioned hereinabove. Such antigens can be detected inthe body fluids using techniques known in the art, such asradioimmunoassays (RIA) or enzyme-linked immunoabsorbant assays (ELISA),wherein antibody reactive with the shed antigen is used to detect thepresence of the antigen in a fluid sample (See, e.g., Uotila et al.,1981, J. Immunol. Methods, 42:11 and S. T. Fayed et al., 1998, DiseaseMarkers, 14(3):155-160). Detection of the shed SCLC antigen is carriedout as described above.

[0115] In yet a further aspect of the invention, monoclonal antibodiesor binding fragments to the SCLC surface glycoprotein are providedlabeled with a detectable moiety, such that they may be packaged andused, for example, in kits, to diagnose or identify cells having theaforementioned antigen. The kits preferably contain an instructionmanual for use of the kit. Non-limiting examples of such labels includefluorophores such as fluorescein isothiocyanate; chromophores,radionuclides, or enzymes. Such labeled antibodies or binding fragmentsmay be used for the histological localization of the antigen, ELISA,cell sorting, as well as other immunological techniques for detecting orquantifying the antigen, and cells bearing the antigen, for example.

[0116] The anti-SCLC antibodies of the present invention exhibit anarrow tumor specificity. In this regard, representative MoAb 109.12reacted with human SCLC antigen but not with human neuroendocrineantigens, human (non-SCLC) lung, human multiple myeloma tissue, humanovarian tissues (fresh ovarian tissue and cell line), human chronicmyelogenous leukemia tissue, and a human B cell tumor. However, theSCLC-specific MoAbs were found to immunoreact with cells from a humanpancreatic cancer and exhibited a distinct pattern of reactivity (FIGS.6A and 6B). In this regard, cell lines from various human canceroustissues or cell lines were tested for binding to the monoclonalantibodies according to the present invention.

[0117] The results of experiments in which the 109.12 and 51.2 MoAbsserved as representative antibodies demonstrated that of threepancreatic tumors tested, two that reacted with the monoclonalantibodies exhibited a different pattern of reactivity, and theantibodies also bound to cell surface antigens of distinct MW (i.e., an˜200 kDa antigen, as well as an antigen of 79-120 kDa on the pancreaticcancer, versus the single antigen of ˜200 KDa on the SCLC cells).Moreover, the SCLC-specific MoAbs did not react with human B cell tumors(IM9 and HT), human breast tissues (fresh breast tissue and cell line)and human prostate tissue. According to this invention, theSCLC-specific MoAbs immunoreact with different epitopes of the sameantigen on pancreatic cancer cells and exhibit narrow tumor specificity.

[0118] Characterization of the SCLC 200 KDa surface antigen shows thatthis antigen is detected by MoAbs 51.2, 109.12 and 37.14 as a singleband by Western Blot analysis (see, for example, FIG. 8B usingrepresentative MoAb 109.12). That the approximately 200 KDa antigen isan integral membrane protein is reflected by its remaining intactfollowing boiling (5 minutes), solubilization with the nonionicdetergent IGEPAL® (Sigma, St. Louis, Mo.) and solubilization with SDS.After immunoaffinity purification of the IGEPAL®-solubilized portion ofthe antigen on a CN-Br-activated Sepharose column coupled to the 109.12monoclonal antibody, the 200 KDa antigen comprises two bands, both ofwhich are between about 75 and 120 MW in size.

[0119] In internalization studies (FIGS. 7A and 7B), the MoAb 51.2 wasshown to internalize into human SCLC cells, but not to internalize intocells of a human neuroendocrine cancer cell line. Such findings have adirect bearing on the target specificity of the monoclonal antibodiesdescribed herein. The internalization results indicate that theantibody(ies) can be conjugated to toxins, which upon binding of theantibody(ies) to the cancerous cells, will deliver the toxin inside thecells and thus will eradicate the cells without compromising the cellsand tissue in the vicinity. The internalization parallels the bindingattributes of the MoAbs, e.g., 51.2, and underscores the highspecificity of the antibodies, particularly the 51.2 MoAb for SCLCcells.

EXAMPLES

[0120] The examples as set forth herein are meant to exemplify thevarious aspects of carrying out the invention and are not intended tolimit the invention in any way.

Example 1

[0121] Materials And Methods

[0122] Sources of Cells

[0123] Cells and cell lines utilized and evaluated in the experimentsdescribed were purchased from the American Type Culture Collection(ATCC), 10801 University Boulevard, Manassas, Va. 20110-2209.Specifically, the cell lines include human SCLC cell lines, i.e., DMS114(ATCC No. CRL-2066), NCI-H209 (ATCC No. HTB-172), NCI-H510A (ATCC No.HTB-184); neuroendocrine cell lines, i.e., SK-N-AS (ATCC No. CRL-2137),Be(2)-/M17 (ATCC No. CRL-2267), MC-IXC (ATCC No. CRL-2270); LnCap(prostate cancer cell line, ATCC No. CRL-1740); Caski (cervical cancercell line, ATCC No. 1550); Namalwa (EBV-transformed tumor cell line,ATCC No. CRL-1432); HT (ATCC No. CRL-2260); IM9 (ATCC No. CCL-159);human myeloma cell lines (U266, OPM, RPMI-1860, KR12 and NCI H929);neuroblastoma cell line (NCI H2106); and the chronic myelogenousleukemic cell line K562 (ATCC No. CCL-243). Fresh clinical specimenssuch as breast cancer, liver carcinoma, ovarian cancer, cervical cancerswere obtained from Stanford University Hospital (CA) or NDRI (MD).

[0124] Preparation Of Cell Membranes

[0125] Cells were lysed following 3 repeated cycles of freeze-thaw.Membranes were prepared from cell lysates following a 30 minutecentrifugation at 2500 rpm. The supernatant, which contained cytosolicprotein and membranes, was further separated by centrifugation at 40,000rpm. Pellets containing the membrane fractions were collected and storedat −20° C.

[0126] For detergent extraction of membrane proteins, cell membranes ata concentration of approximately 10 mg/ml were diluted 1:1 in IGEPAL®CA-630 (a nonionic detergent formerly known as Nonidet P-40), at 0.312%in PBS (final detergent concentration of 0.156%) and incubated on icefor 1 hour. The preparations were centrifuged for 1 hour at 4° C. at16,000×g. The soluble portion (supernatant) was subjected to SDS-PAGEand detected with MoAb by Western Blot (see FIG. 8B).

[0127] Mice and Immunization

[0128] Balb/c and SCID (6-8 week old female) mice were purchased fromTaconic (NY). Mice were immunized with a pool of human SCLC cells (i.e.,DMS114, NCI-H209 and NCI-H510A). About 5×10⁵-5×10⁶ total cells (in 50 μlcontaining Ribi adjuvant, 50% v/v) were injected into the right footpad.In the left footpad, mice were injected with a pool of humanneuroendocrine cell lines (SKON-AS, MC-IXC and Be(2)-/M17), in 50 μlcontaining Ribi adjuvant, 50% v/v). The mice were boosted 14 days laterwith a pool of SCLC cells (5×10⁴-5×10⁵ total). The right popliteal lymphnode was removed and the extracted cells were fused with murine myelomaSp2/0 cells three days after the second immunization.

[0129] Generation Of B Cell Hybridomas

[0130] Monoclonal antibodies specific to SCLC were produced byconventional methods. Spleen cells were fused with murine myeloma sp2/0and seeded into 96 well-plates at a final concentration of 1×10⁴cells/well.

[0131] Cellular ELISA

[0132] Supernatants from hybridoma cultures were screened for directbinding to membrane preparations extracted from a pool of SCLC celllines and compared with background binding to control neuroendocrinecell lines, using an ELISA assay. SCLC and neuroendocrine cells (1×10⁵cells/well) were dispensed into a 96-well tissue culture plate (Costar,Cambridge, Mass.) and incubated with hybridoma supernatant (50 μl/well)for 30 minutes at room temperature (RT). The cells were then washedthree times with phosphate buffered saline, pH 7.4 (PBS), (Gibco BRL).Next, biotinylated rabbit anti-mouse (gamma) solution (0.5 μg/ml) wasadded to the wells (50 μg per well). Following a 1-hour of incubationand extensive washes as above, streptavidin-conjugated horseradishperoxidase (Zymed, CA), diluted to 1:10,000 in blocking buffer, wasadded to the wells and a 30-minute incubation followed. Substrate wasadded (100 μl/well of tetramethylbenzidine (DAKO Corporation,California) and the plates were incubated for 30 minutes at RT. Thereaction was stopped using 100 μl/well of 2N H₂SO₄. The plates were readusing an automated ELISA Plate Reader at a wavelength of 450 nm.

[0133] Gel Electrophoresis And Western Blotting

[0134] SDS-polyacrylamide gels (8%) were prepared according to standardprotocols and used for Western blotting. Membrane preparations orhomogenates (50 μg/lane) (e.g., tumor cells lines, fresh normal humantissues, and freshly excised human tumors) were loaded onto the aboveSDS gels and fractionated. Blots were incubated in a blocking buffercontaining 10% BSA in TBST buffer (20 mM Tris-base, 137 mM NaCl {pH7.6}, and 0.05% Tween 20) for 1-hour, followed by incubation in theprimary MoAb (0.1 μg/ml) for 1-2 hours. After washing 3 times in TBSTbuffer, blots were incubated for 30 minutes with a horse radishperoxidase (HRP)-conjugated secondary antibody (0.05 μg/ml). The blotswere developed with the ECL™ (Amersham Pharmacia Biotech, Piscataway,N.J.) Western Blot chemiluminescent detection reagent according to themanufacturer's instructions.

[0135] Evaluation of Carbohydrates in Glycoprotein(s) Recognized bySCLC-specific MoAbs

[0136] Blots of membranes from SCLC prepared as described above aretested with a panel of lectins (e.g., WGA, Triticum vulgar or wheatgerm; PNA, Arachis hypogaea or peanut; BPA, Bauhinia purpurea or camel'sfoot tree; SBA, Glycine max or soybean; MPA, Maclura pomifera or osageorange; UEA-l, Ulex europaeus or gorse; and ConA, Concanavaliaensiformis or jack bean) to determine the presence of specific sugarresidues. As an alternative, radiolabeled lysates are preabsorbed withvarious lectin-agarose beads (Vector Labs, Burlingame, Calif.), and thenincubated with the monoclonal antibodies of the present invention. Theantigen (bound to the MoAbs) is immunoprecipitated by incubation of thesupernatant with beads coated with anti-mouse Ig, and then fractionatedby SDS-PAGE. Elimination of the antigen from the immunoprecipitatedmaterial will indicate that the antigen binds to the pre-clearing lectinand therefore expresses the respective sugar specificity.

[0137] Another alternative protocol for carbohydrate identificationinvolves the inhibition of monoclonal antibody binding to antigenthrough competition with lectin-specific antibodies. The lectin-specificantibodies are available in either a non-conjugated form or conjugatedto biotin or to fluorescein. The choice availability allows performanceof the assay using various methods. Commercial kits (Vector Labs)containing the lectins and their controls are available. The lectinscreening kits are designed to provide the investigator with a panel ofseven lectins or lectin conjugates. The lectins have been selected tooffer a variety of sugar specificities and are of the same high qualityas the reagents offered individually.

[0138] Complement-Mediated Cytotoxicity

[0139] Tumor cells are incubated in RPMI+10% FBS at 1×10⁵ cells/well in96 well plates. Protein-G purified SCLC-specific MoAbs or isotypecontrol antibody are added at 1 μg/ml to 10 μg/ml in the presence ofhuman complement for 1 hour at 37° C. and 5% CO₂. Controls platescontain tumor cells incubated with antibody alone, complement alone, 1%SDS, complete medium alone, and 2 μg/ml of PHA. Plates are pulsed with 1μCi/well of ³H-Thymidine (Dupont-NEN) for 12-16 hours at 37° C.,harvested and read on a Wallac Trilux MicroBeta counter. Percent lysisis calculated as: 100−{[mean counts/mean spontaneous proliferation(CM)]×100}. An adjusted percent lysis is calculated as [Mean percentlysis/mean maximum lysis]×100.

[0140] In Vivo Eradication of Tumor Cells

[0141] SCLC or control tumor cells are injected intraperitoneally (i.p.)into C.B-17 SCID beige mice (Taconic, N.Y.). Each mouse receives 1×10⁶cells washed and resuspended in PBS. After ten days, the mice areinjected i.p. with 200 μg Protein-G purified, SCLC-specific MoAbs, orisotype-matched control antibody, 3 times a week for 3 weeks. Miceincapacitated by the large tumor are euthanized. Mice are monitored forascites growth and results are plotted as a percentage of survival.

[0142] Internalization of Antibody

[0143] Studies are conducted to measure the internalization of MoAbswithin SCLC cells. The MoAbs are conjugated to the ricin A chain toxinto form an immunotoxin. Internalization will be assessed by determiningto what extent the cancer cells are killed by ricin A chain toxin (U.S.Pat. No. 5,491,088 to I. Hellstrom et al., issued Feb. 13, 1996).

Example 2

[0144] Positive Hybridomas Produce Monoclonal Antibodies thatSpecifically bind to SCLC

[0145] B cell hybridomas established from immunized mice as described inExample 1 were screened by cellular ELISA with a pool of human SCLC celllines (i.e., DMS 114, NCI-H209, NCI-H510A) compared with a pool of humanneuroendocrine cell lines (SK-N-AS, MC-IXC, Be (2)-M17). Elevenhybridomas out of a pool of 170 were selected based on their productionof monoclonal antibodies having the ability to specifically bind toSCLC. Limiting dilution cultures (1 cell per well) were set in up inorder to establish clonal B cell hybridomas. The resultant sub-cloneswere screened as described above. Twelve representative sub-clonesexhibiting the highest mean values for absorbance against the SCLC cellswere selected for further testing. Values were adjusted by subtractingthe mean O.D. of the neuroendocrine cells from the SCLC cells (FIG. 1).The selected monoclonal antibody-producing B cell hybridomas wereisotyped to establish the Ig gene usage (i.e., immunoglobulin isotype ofthe produced antibody) as well as to validate clonality of thesub-cloned hybridoma.

[0146] Example 3

[0147] MoAbs React with Antigens Present in the Cell Membranes of SCLC

[0148] The selected panel of monoclonal antibodies was tested by FACSfor staining of SCLC cells compared with control (neuroendocrine ormultiple myeloma) cell lines. Four MoAbs, 26.1, 141.7, 92.7, and 37.14,exhibited a stronger staining of SCLC cells, when compared with thecontrol cell lines. Western blotting performed under reducing conditionswas used to further elucidate the specificity of the selected MoAbs.MoAbs 51.2 and 109.12 detected an antigen with a MW of about 200 KDa inthe lanes containing the pool of SCLC cell lines (DMS114, NCI-H209, andNCI-H510A), (FIGS. 2 and 3). The appearance of a single fuzzy bandindicates that the molecule recognized by the MoAbs is a single chainglycoprotein.

[0149] The control cell membrane preparations isolated from a pool ofneuroendocrine (SK-N-AS, MC-IXC, and Be (2)-M17) and multiple myeloma(RPMI 8226 and U266) cell lines, respectively, were not stained by theMoAb 109.12. A significantly fainter band appeared in the neuroendocrinelane and was absent in the MM lane for the MoAb 51.2. This observationmay indicate that the recognized SCLC antigen is over-amplified in theSCLC cells, compared with the closely related neuroendocrine cells. Theantigen does not seem to be expressed by MM cells, which are moredevelopmentally distant from the SCLC cells.

[0150] Monoclonal antibody 37.14 reacted with an antigen having a lowermolecular weight (between 35-50 kDa). The latter antigen appeared tocomprise two fragments or chains, which are closely related in theirmass (FIG. 4). Interestingly, MoAb 26.1 plainly recognized aSCLC-specific cell surface molecule, but failed to react with SCLCmembranes in a Western Blot performed under reducing conditions. Thisresult may indicate that the epitope recognized by MoAb 26.1 is aconformational epitope, which is destroyed by linearization treatment inthe presence of SDS.

Example 4

[0151] Immunohistochemical Analysis

[0152] For the immunohistochemical analyses performed on normal humanlung tissue and SCLC diseased tissue (FIGS. 9A-9D), the followingmethods and materials were employed:

[0153] Tissue Source: Histologically normal human tissues and tumorswere obtained from surgical and autopsy specimens and prepared forimmunohistochemical analysis (IMPATH, Los Angeles, Calif.). Freshtissues were embedded in OCT compound (Miles Laboratories, Inc.,Naperville, Ill.) and snap-frozen in isopentane cooled by liquidnitrogen. Specimens were stored at−80° C. until needed. Tissue was cutat 5 microns, placed on positively-charged slides, and air-dried. Ahuman MM cell line (U266) served as a positive control, and two B celllines (IM9, HT), were used as negative controls.

[0154] Reagents: The 51.2 MoAb was used as test antibody in theimmunohistochemical analyses. The negative reagent control, murine IgG1,was purchased from DAKO Corporation (Carpinteria, Calif.). Theantibodies were diluted to working concentrations with Primary AntibodyDiluent (Research Genetics, Huntsville, Ala.).

[0155] Immunohistochemistry: Studies performed using an indirectperoxidase-conjugated immunohistochemical detection technique; the DAKOEnvision+™ System (DAKO Corporation), according to the manufacturer'sinstructions. Cryostat-cut sections were removed from the−80° C.freezer, and air-dried for 30 minutes. Slides were fixed in acetone for5 minutes at 4 C. and washed in Phosphate Buffered Saline (PBS; Amresco,Solon, Ohio) at pH 7.2. Endogenous peroxidase activity was blocked witha 5-minute hydrogen peroxide solution, followed by PBS washes.

[0156] The slides were incubated with the 51.2 monoclonal antibody, orthe isotype-matched control antibody, for 30 minutes at roomtemperature, followed by PBS washes. Next, the slides were incubatedwith an anti-mouse antibody conjugated to a peroxidase-labeled dextranpolymer for 30 minutes at room temperature and then washed in PBS. Theperoxidase reaction was visualized by incubating for 5±1 minutes with3,3′-diaminobenzidine-tetrahydrochloride solution. The slides werethoroughly washed with tap water, counterstained with a modified Harrishematoxylin (American Master Tech. Scientific Inc., Lodi, Calif.),dipped in 0.25% acid alcohol, blued in 0.2% ammonia, dehydrated throughgraded alcohols, cleared in xylene, and coverslipped.

[0157] Controls: Positive control tissue sections were derived from afrozen cell block prepared from the U266 cell line. Negative controlsections were derived from frozen cell blocks prepared from IM9 and HTcell lines. For the negative reagent control, the primary antibody wassubstituted with an isotype-matched control antibody at the sameantibody concentration as the test article. The negative control sectionrefers to the tissue section to which the isotype control antibody wasapplied.

[0158] Interpretation of Slides: Interpretation of stained slides wasperformed by microscopic examination. A morphologic review of the tissueon the slide determined whether an adequate amount of tissue waspresent, and whether the designated tissue was appropriatelyrepresented. Samples failing to meet the above standards were rejectedfrom the analysis. The staining intensity of the test article was judgedrelative to the intensity of a control slide. Staining of the sectionlabeled with the negative reagent control was considered “background.”

[0159] Fixation Analyses: Analysis was performed using the positive andnegative control cell lines, U266 and IM9, respectively. Of thefixatives evaluated (unfixed, acetone, ethanol, methanol/acetone, and10% neutral buffered formalin), acetone for 5 minutes at 4° C. resultedin the best combination of morphological preservation and stainingintensity.

[0160] The contents of all patent applications, issued patents,published articles and references, and textbooks as cited herein arehereby incorporated by reference in their entirety to more fullydescribe the state of the art to which the present invention pertains.

[0161] As various changes can be made in the above compositions andmethods without departing from the scope and spirit of the invention, itis intended that all subject matter contained in the above description,shown in the accompanying drawings, or defined in the appended claims beinterpreted as illustrative, and not in a limiting sense.

What is claimed is:
 1. A monoclonal antibody, or binding fragmentthereof, which binds specifically to an antigen on the surface of smallcell lung cancer (SCLC) cells, the antigen being characterized in that(i) it is a single polypeptide having a molecular weight of about 200kDa as determined by SDS-PAGE under reducing conditions; (ii) it isabsent from human multiple myeloma cells; and (iii) it is glycosylated.2. The monoclonal antibody, or binding fragment thereof, according toclaim 1, which is produced by a hybridoma cell line deposited at theAmerican Type Culture Collection (ATCC) and having ATCC Accession Numberselected from the group consisting of ATTC Accession No. PTA-2360 (MoAb51.2), ATCC Accession No. PTA-2358 (MoAb 37.14) and ATCC Accession No.PTA-2357 (MoAb 109.12).
 3. The monoclonal antibody, or binding fragmentthereof, according to claim 1, which binds specifically to an antigen onthe surface of small cell lung cancer (SCLC) cells, the antigen beingfurther characterized in that (iv) it is absent from neuroendocrinecells.
 4. The monoclonal antibody, or binding fragment thereof,according to claim 3, which is produced by a hybridoma cell linedeposited at the American Type Culture Collection (ATCC) and having ATCCAccession Number PTA-2357 (MoAb 109.12).
 5. A monoclonal antibody, orbinding fragment thereof, which binds specifically to an antigen on thesurface of small cell lung cancer (SCLC) cells, the antigen beingcharacterized in that (i) it has a molecular weight of about 35 kDa toabout 50 kDa as determined by SDS-PAGE under reducing conditions; (ii)it is absent from human multiple myeloma cells and neuroendocrine cells;and (iii) it is glycosylated.
 6. The monoclonal antibody, or bindingfragment thereof, according to claim 5, which is produced by a hybridomacell line deposited at the American Type Culture Collection (ATCC) andhaving ATCC Accession Number PTA-2358 (MoAb 37.14).
 7. A monoclonalantibody, or binding fragment thereof, which (i) binds specifically to aconformational epitope of an antigen on the surface of small cell lungcancer (SCLC) cells, and (ii) is produced by a hybridoma cell linedeposited at the American Type Culture Collection (ATCC) having ATCCAccession No. PTA-2359 (MoAb 26.1).
 8. A monoclonal antibody, or bindingfragment thereof, selected from the group consisting of (i) a monoclonalantibody produced from the hybridoma cell line deposited at the AmericanType Culture Collection (ATCC) having ATCC Accession No. PTA-2358 (MoAb37.14), a monoclonal antibody produced from the hybridoma cell linedeposited at the American Type Culture Collection (ATCC) having ATCCAccession No. PTA-2360 (MoAb 51.2), or a hybridoma cell line depositedat the American Type Culture Collection (ATCC) having ATCC Accession No.PTA-2357 (MoAb 109.12), which antibodies bind to a cell surfaceglycoprotein antigen on human small cell lung cancer cells, and (ii)antibodies that are capable of binding to the same antigenic determinantas do the monoclonal antibodies produced by the hybridoma cell linesdeposited at the American Type Culture Collection (ATCC) having ATCCAccession No. PTA-2358 (MoAb 37.14), having ATCC Accession No. PTA-2360(MoAb 51.2), or having ATCC Accession No. PTA-2357 (MoAb 109.12).
 9. Amonoclonal antibody, or binding fragment thereof, selected from thegroup consisting of a monoclonal antibody produced from the hybridomacell line deposited at the American Type Culture Collection (ATCC)having ATCC Accession No. PTA-2360 (MoAb 51.2), a hybridoma cell linedeposited at the American Type Culture Collection (ATCC) having ATCCAccession No. PTA-2357 (MoAb 109.12), a hybridoma cell line deposited atthe American Type Culture Collection (ATCC) having ATCC Accession No.PTA-2358 (MoAb 37.14) and a hybridoma cell line deposited at theAmerican Type Culture Collection (ATCC) having ATCC Accession No.PTA-2359 (MoAb 26.1).
 10. The monoclonal antibody, or binding fragmentthereof, according to claim 8, wherein the cell surface glycoproteinantigen is present on human small cell lung cancer cells and is absentfrom human multiple myeloma cells.
 11. The monoclonal antibody, orbinding fragment thereof, according to claim 8, wherein the cell surfaceglycoprotein antigen recognized by the monoclonal antibody or bindingfragment thereof is present on the surface of human small cell lungcancer cells and is a single glycosylated polypeptide having a molecularweight of about 200 kDa as determined by SDS-PAGE under reducingconditions.
 12. The monoclonal antibody or binding fragment thereof,according to any one of claims 1, 5, or 9, wherein the binding fragmentis selected from the group consisting of Fab fragments, F(ab)₂fragments, Fab′ fragments, F(ab′)₂ fragments, Fd fragments, Fd′fragments and Fv fragments.
 13. An anti-idiotypic antibody which mirrorsthe binding site of the antibody according to claim
 9. 14. A hybridomacell line which produces a monoclonal antibody which binds specificallyto an antigen on the surface of small cell lung cancer (SCLC) cells, theantigen being characterized in that (i) it is a single polypeptidehaving a molecular weight of about 200 kDa as determined by SDS-PAGEunder reducing conditions; (ii) it is absent from human multiple myelomacells; and (iii) it is glycosylated.
 15. The hybridoma cell lineaccording to claim 14, which is deposited at the American Type CultureCollection (ATCC) under ATCC Accession No. PTA-2360 (MoAb 51.2).
 16. Thehybridoma cell line according to claim 14, which is deposited at theAmerican Type Culture Collection (ATCC) under ATCC Accession No.PTA-2357 (MoAb 109.12).
 17. The hybridoma cell line according to claim14, which is deposited at the American Type Culture Collection (ATCC)under ATCC Accession No. PTA-2358 (MoAb 37.14).
 18. The hybridoma cellline according to claim 14, wherein the antigen on the surface of smallcell lung cancer (SCLC) cells which is bound by the produced monoclonalantibody, or binding fragment thereof, is further characterized in that(iv) it is absent from neuroendocrine cells.
 19. The hybridoma cell lineaccording to claim 18, which is deposited at the American Type CultureCollection (ATCC) under ATCC Accession No. PTA-2357 (MoAb 109.12).
 20. Ahybridoma cell line which produces a monoclonal antibody which bindsspecifically to an antigen on the surface of small cell lung cancer(SCLC) cells, the antigen being characterized in that (i) it has amolecular weight of about 35 kDa to about 50 kDa as determined bySDS-PAGE under reducing conditions; (ii) it is absent from humanmultiple myeloma cells and neuroendocrine cells; and (iii) it isglycosylated.
 21. The hybridoma cell line according to claim 20, whichis deposited at the American Type Culture Collection (ATCC) under ATCCAccession No. PTA-2358 (MoAb 37.14).
 22. An antibody-recognized surfaceantigen present on human small cell lung cancer cells and having thecharacteristics of (i) being a single polypeptide having a molecularweight of about 200 kDa as determined by SDS-PAGE under reducingconditions; (ii) being absent from human multiple myeloma cells; (iii)being absent from or underexpressed on neuroendocrine cells; and (iv)being glycosylated.
 23. An antibody-recognized surface antigen presenton human small cell lung cancer cells, the antigen having thecharacteristics of (i) having a molecular weight of about 35 kDa toabout 50 kDa as determined by SDS-PAGE under reducing conditions; (ii)being absent from human multiple myeloma cells and human neuroendocrinecells; and (iii) being glycosylated.
 24. The antibody-recognized surfaceantigen according to claim 22, wherein the antibody that binds to theantigen is a monoclonal antibody produced by a hybridoma cell linedeposited at the American Type Culture Collection (ATCC) and selectedfrom the group consisting of the hybridoma cell line having ATCCAccession No. PTA-2358 (MoAb 37.14), the hybridoma cell line having ATCCAccession No. PTA-2360 (MoAb 51.2) and the hybridoma cell line havingATCC Accession No. PTA-2357 (MoAb 109.12).
 25. The antibody-recognizedsurface antigen according to claim 23, wherein the antibody that bindsto the antigen is a monoclonal antibody produced by a hybridoma cellline deposited at the American Type Culture Collection (ATCC) and havingATCC Accession No. PTA-2358 (MoAb 37.14).
 26. A method of inhibiting orkilling small cell lung cancer (SCLC) cells, comprising: providing to apatient in need thereof the monoclonal antibody, or binding fragmentthereof, according to any one of claims 1, 5, or 9, under conditions andin an amount sufficient for the binding of the monoclonal antibody, orbinding fragment thereof, to the SCLC cells, thereby causing inhibitionor killing of the SCLC cells by the immune cells of the patient.
 27. Themethod according to claim 26, further wherein the monoclonal antibody isconjugated with a cytotoxic moiety.
 28. The method according to claim27, wherein the cytotoxic moiety is a chemotherapeutic agent, aphotoactivated toxin, or a radioactive agent.
 29. The monoclonalantibody, or binding fragment thereof, according to claim 1, claim 5, orclaim 9 bound to a solid matrix.
 30. A method of localizing small celllung cancer (SCLC) cells in a patient, comprising: (a) administering tothe patient a detectably-labeled monoclonal antibody, or bindingfragment thereof, according to any one of claims 1, 5, or 9; (b)allowing the detectably-labeled monoclonal antibody, or binding fragmentthereof, to bind to the SCLC cells within the patient; and (c)determining the location of the labeled monoclonal antibody or bindingfragment thereof, within the patient.
 31. A method of detecting thepresence and extent of small cell lung cancer in a patient, comprising:determining the level of the antigen according to any of claims 22 to 25in a sample of bodily fluid from the patient and correlating thequantity of the antigen with the presence and extent of the small celllung cancer disease in the patient.
 32. A method of monitoring theeffectiveness of therapy for small cell lung cancer disease, comprising:periodically measuring changes in the level of the antigen according toany of claims 22 to 25 in a body fluid sample taken from a patientundergoing the therapy, and correlating the change in level of theantigen with the effectiveness of the therapy, wherein a lower level ofantigen determined at a later time point relative to the level ofantigen determined at an earlier time point during the course of therapyindicates effectiveness of the therapy for small cell lung cancerdisease.
 33. A method of diagnosing the presence of small cell lungcancer in a patient, comprising: (a) measuring the levels of the antigenaccording to any of claims 22 to 25 in cells, tissues, or body fluids ofthe patient; and (b) comparing the measured levels of the antigen of (a)with levels of the antigen in cells, tissues, or body fluids from anormal human control, wherein an increase in the measured levels of theantigen in the patient versus the normal control is associated with thepresence of small cell lung cancer.
 34. A method of imaging small celllung cancer in a patient, comprising administering to the patient theantibody according to any one of claims 1, 5, or 9, wherein the antibodyis detectably labeled with paramagnetic ions or with a radioisotope. 35.A pharmaceutical composition comprising the monoclonal antibody, orbinding fragment thereof, according to any one of claims 1, 5, or 9, anda pharmaceutically acceptable carrier, excipient, or diluent.
 36. Thepharmaceutical composition according to claim 35, wherein the monoclonalantibody, or binding fragment thereof, recognizes an antigen present onthe surface of human small cell lung cancer cells, but not on normallung cells, or on human multiple myeloma cells.
 37. A pharmaceuticalcomposition comprising a monoclonal antibody, or binding fragmentthereof, wherein the antibody (a) is selected from the group consistingof (i) a monoclonal antibody produced from the hybridoma cell linedeposited at the American Type Culture Collection (ATCC) and having ATCCAccession No. PTA-2360 (MoAb 51.2), (ii) a monoclonal antibody producedfrom the hybridoma cell line deposited at the American Type CultureCollection (ATCC) and having ATCC Accession No. PTA-2357 (MoAb 109.12);(iii) a monoclonal antibody produced from the hybridoma cell linedeposited at the American Type Culture Collection (ATCC) and having ATCCAccession No. PTA-2358 (MoAb 37.14), (iv) a monoclonal antibody producedfrom the hybridoma cell line deposited at the American Type CultureCollection (ATCC) and having ATCC Accession No. PTA-2359 (MoAb 26.1);wherein the monoclonal antibodies (i)-(iv) bind to a cell surfaceantigen on human small cell lung cancer cells; (b) is an antibody thatis capable of binding to the same antigenic determinant as does themonoclonal antibody, or a binding fragment thereof, produced by ahybridoma cell line deposited at the American Type Culture Collection(ATCC) under ATCC Accession No.PTA-2360 (MoAb 51.2), or produced by ahybridoma cell line deposited at the American Type Culture Collection(ATCC) under ATCC Accession No. PTA-2357 (MoAb 109.12), or produced by ahybridoma cell line deposited at the American Type Culture Collection(ATCC) under ATCC Accession No. PTA-2358 (MoAb 37.14), or produced by ahybridoma cell line deposited at the American Type Culture Collection(ATCC) under ATCC Accession No. PTA-2359 (MoAb 26.1); and apharmaceutically acceptable carrier, excipient, or diluent.
 38. Themonoclonal antibody according to any one of claims 1, 5, or 9, labeledwith a detectable moiety.
 39. The monoclonal antibody according to claim38, wherein the detectable moiety is selected from the group consistingof a fluorophore, a chromophore, a radionuclide, a chemiluminescentagent, a bioluminescent agent and an enzyme.
 40. A method of inhibitingor killing pancreatic cancer cells, comprising: providing to a patientin need thereof the monoclonal antibody, or binding fragment thereof,according to any one of claims 1, 5, or 9, under conditions and in anamount sufficient for the binding of the monoclonal antibody, or bindingfragment thereof, to the SCLC cells, thereby causing inhibition orkilling of the SCLC cells by the immune cells of the patient.
 41. Themethod according to claim 40, further wherein the monoclonal antibody isconjugated with a cytotoxic moiety.
 42. The method according to claim41, wherein the cytotoxic moiety is a chemotherapeutic agent, aphotoactivated toxin, or a radioactive agent.
 43. A method of localizingpancreatic cancer cells in a patient, comprising: (a) administering tothe patient a detectably-labeled monoclonal antibody, or bindingfragment thereof, according to any one of claims 1, 5, or 9; (b)allowing the detectably-labeled monoclonal antibody, or binding fragmentthereof, to bind to the pancreatic cancer cells within the patient; and(c) determining the location of the labeled monoclonal antibody orbinding fragment thereof, within the patient.
 44. The method accordingto claim 43, wherein the detectable label is selected from the groupconsisting of a fluorophore, a chromophore, a radionuclide, achemiluminescent agent, a bioluminescent agent and an enzyme.
 45. Amethod of imaging pancreatic cancer in a patient, comprisingadministering to the patient the antibody according to any one of claims1, 5, or 9, wherein the antibody is detectably labeled with paramagneticions or with a radioisotope.
 46. A method of diagnosing or detectingpancreatic tumor cells or cancer cells in a patient, comprising:incubating the monoclonal antibody, or binding fragment thereof,according to any one of claims 1, 5, or 9 with pancreatic cells from apatient; and detecting the binding of the monoclonal antibody to thepancreatic cells, thereby diagnosing or detecting pancreatic tumor cellsor cancer cells in the patient.
 47. The method according to claim 46,wherein the monoclonal antibody is MoAb 109.12 having ATCC Accession No.PTA-2357.
 48. The method according to claim 46, wherein the monoclonalantibody is labeled with a detectable label selected from the groupconsisting of a fluorophore, a chromophore, a radionuclide, achemiluminescent agent, a bioluminescent agent and an enzyme.
 49. Apharmaceutical composition comprising the monoclonal antibody, orbinding fragment thereof, according to any one of claims 1, 5, or 9, anda pharmaceutically acceptable carrier, excipient, or diluent, whereinthe monoclonal antibody, or binding fragment thereof, recognizes anantigen present on the surface of human pancreatic cancer cells.